Colloidal silver composition having antimicrobial properties

ABSTRACT

Colorless composition comprising silver particles and water, wherein said particles comprise an interior of elemental silver and an exterior of ionic silver oxide, wherein the silver particles are present in the water at a level of about 5-40 ppm, and wherein the composition manifests significant antimicrobial properties. Methods of use of the composition are described. The composition can be incorporated into a hydrogel with essentially no loss of antimicrobial properties.

The present application is a continuation-in-part of application Ser.No. 09/946,834, filed Sep. 4, 2001, which is a continuation ofapplication Ser. No. 09/323,310, filed Jun. 1, 1999, now U.S. Pat. No.6,214,299. To the extent permitted these applications are incorporatedherein by reference. The present application claims priority toprovisional application 60/475,657, filed Jun. 3, 2003, and incorporatedby reference herein.

AREA OF THE ART

The present invention generally relates to colloidal silver, and moreparticularly to a composition of colloidal silver and a method for usingsaid composition as an agent against organisms harmful to the health ofhumans.

DESCRIPTION OF THE PRIOR ART

It is well known that certain preparations of silver have germicidalproperties. Silver was employed as a germicide and an antibiotic beforemodern antibiotics were developed. In previous centuries, users wouldshave silver particles into their drinking water, or submerge wholesilver pieces in the drinking water, for the purpose of ingesting thesilver by drinking the water. It seems likely that the practice ofeating with silver utensils (i.e., silverware) resulted from a belief inthe healthful properties of silver.

There may be many reasons why administering silver suspended in solutionwould enhance an individual's health. It is possible that such asolution operates to inhibit the growth of bacteria, viruses, and otherunwanted organisms, as well as eradicating such existing bacteria,viruses, and other organisms. It is also possible that a solution ofsilver can have an anti-inflammatory effect, sufficient to reducesymptoms of asthma.

The present invention describes the use of a silver composition in waterto treat certain human ailments. An embodiment of the invention is asilver composition comprising small particles of silver which comprisean interior of metallic silver and an exterior of ionic silver whichparticles are suspended in water. A preferred embodiment of theinvention is a silver composition comprising particles of silver whereinmore than 50% of the number of particles are less than 0.015 micrometersin size and the particles are colloidally suspended in water.

SUMMARY OF THE INVENTION

The present invention is generally directed to the use of silver, at alevel of 5 to 40 ppm in water, to kill or to disable microorganismswhich are hazardous to human beings. The present invention specificallyis directed to compositions comprising silver particles, said particlescomprising an interior of elemental silver and an exterior of ionicsilver oxide, and water, wherein the silver particles are placed incolloidal suspension in the water at a level of 5-40 ppm total silver.An embodiment of the present invention comprises silver particles inwater, at a concentration of 5-40 ppm, wherein more than 50% of thesilver particles have a maximum dimension less than 0.015 micrometers.The composition of silver in water of this invention is an effectiveantimicrobial agent. This invention is directed to silver compositions,of 5-40 ppm silver in water, which are effective antimicrobial agents,and to methods of using said silver compositions as antimicrobialagents.

A preferred embodiment of the present invention is directed tocompositions of silver in water made using a modification of the deviceand methods described in U.S. Pat. No. 6,214,299, which is a parent ofthe instant application and is incorporated herein by reference.

The device and process of U.S. Pat. No. 6,214,299 have been modified andimproved to provide the silver composition of the present invention.Essentially, the eight-silver/one common electrode device as disclosedin the patent has been modified and scaled to fit a 75-gallon waterchamber. To start the process approximately 70 gallons of high puritywater are placed in the chamber. To this is added approximately fivegallons of silver composition produced in a prior production run. Thisis necessary because the high purity water is insufficiently conductivefor the process to occur properly. The water chamber is equipped with anair input that allows a stream of air bubbles to be streamed through theliquid during the processing. It has been discovered that this approachgives improved mixing as compared to the impeller mixer described in thepatent.

The electrode device is operated at approximately ten thousand voltsalternating current (with each silver electrode having an individualvoltage supply) as described in the patent. It has been found thatvoltages significantly lower than this produce a composition with largerparticles not having the optimal properties described herein. Voltagessignificantly higher tend to produce a solution with significant ionicsilver dissolved therein. The present composition comprises in excess of97% metallic silver with essentially no free ionic silver in solution.

The silver concentration is determined according to the methodsexplained below. Essentially, the device is operated continuously andsamples are analyzed until the desired silver concentration is attained.The 10 ppm composition requires approximately one and one half days ofoperation. The 22 ppm solution requires approximately three days, andthe 32 ppm composition requires approximately six days. The rate of theprocess appears to slow as the higher concentrations are attained.Higher concentrations take a prohibitively long time with the ultimatehighest concentration being about 50 ppm, at least within the currentparameters.

The compositions all have the size characteristics described below andunlike conventional silver compositions are completely colorless andstable to light and temperature changes without use of any additives.The compositions are unreactive towards added hydrogen peroxide.

Hydrogen peroxide, a know disinfecting agent, has been found to have asynergistic interaction with the inventive silver composition. Hydrogenperoxide is available in concentration of 30% wght/v (% weight pervolume or weight percent) or higher. Although the higher concentrationsare usable, the preferred concentrations are in the range of 1 to 5%wght/v.

A preferred embodiment of the present invention is directed tocompositions comprising 5 to 40 ppm silver, said silver being primarilyelemental silver, 1 to 3 wght % hydrogen peroxide, and water. Apreferred embodiment of the present invention is the use, and method ofuse, of compositions comprising 10 to 40 ppm silver and 1 to 3 wght %hydrogen peroxide in water as antimicrobial agents.

Although a large number of tests employing the colloidal silver solutionalone are presented below, it is also demonstrated that certain vehiclescan significantly improve the results obtainable with the silver.Specifically it has been found that formulating the aqueous silvercolloid as a semi-solid hydrogel significantly enhances it efficacy.Hydrogels are hydrophilic gels produced by adding certain hydrophilicorganic polymers to an aqueous solution—in this case a solutioncontaining the inventive colloidal silver solution. As would beexpected, the hydrogel improves the retention of the silver on a surfacesuch as a wound. For wound care a hydrogel also has the significantadvantage of protecting the tissues surrounding the wound and preventingdesiccation, which factors enhance wound healing. Most significantly,the hydrogel does not interfere with the antimicrobial properties of thecolloidal silver nor does it appear to significantly slow the diffusionof the active silver colloids.

DETAILED DESCRIPTION OF THE INVENTION

The following description is provided to enable any person skilled inthe art to make and use the invention and sets forth the best modescontemplated by the inventor of carrying out his invention. Variousmodifications, however, will remain readily apparent to those skilled inthe art, since the general principles of the present invention have beendefined herein specifically to provide an improved colloidal silverproduct with significant abilities to kill human pathogens both in vivoand in vitro.

Generally, the present invention represents a novel approach to killingor disabling microorganisms which are hazardous to human beings by theuse of silver particles in water, at a concentration of 5 to 40 ppmsilver. Depending upon the application, the silver composition may beused internally or externally. Depending on the application, the silvercomposition may also contain hydrogen peroxide.

Preferred Embodiments

Non-limiting preferred embodiments are presented in the following:

A composition comprising silver particles, colloidally suspended inwater, wherein the total content of silver is between 5 and 40 ppm,which composition kills or disables microorganisms which are hazardousto the human body.

A composition comprising silver particles, colloidally suspended inwater, wherein the total content of silver is about 10±2 ppm, whichcomposition kills or disables microorganisms which are hazardous to thehuman body.

A composition comprising silver particles, colloidally suspended inwater, wherein the total content of silver is about 22±2 ppm, whichcomposition kills or disables microorganisms which are hazardous to thehuman body.

A composition comprising silver particles, colloidally suspended inwater, wherein the total content of silver is about 32±3 ppm, whichcomposition kills or disables microorganisms which are hazardous to thehuman body.

It will be appreciated that specifying the total amount of silver in acomposition of particles does not completely specify the material. Asthe particles comprising the composition are made smaller, a givenconcentration of silver will represent a larger number of particles. Inaddition, the total surface area for a given silver concentration willincrease. Therefore, particles size and range of particle size is animportant parameter for defining an effective inventive composition.

A further class of embodiments is any of the above-describedcompositions, wherein more than 50% of the silver particles have amaximum dimension less than 0.015 micrometers.

A further class of embodiments is any of the above-describedcompositions, wherein more than 75% of the silver particles have amaximum dimension less than 0.015 micrometers.

A further class of embodiments is any of the above-describedcompositions, wherein more than 90% of the silver particles have amaximum dimension less than 0.02 micrometers.

A further class of embodiments is any of the above-describedcompositions, wherein more than 75% of the silver particles have aminimum dimension greater than 0.005 micrometers.

A further class of embodiments is any of the above-describedcompositions, wherein more than 90% of the silver particles have aminimum dimension greater than 0.005 micrometers.

A further class of embodiments is any of the above-describedcompositions, wherein the silver particles comprise both silver in thezero-valent, that is, metallic, oxidation state [Ag(0)] and a coating ofsilver in an ionic oxidation selected from the group consisting ofAg(I), Ag(II), and Ag(III).

A further class of embodiments is any of the above-describedcompositions, wherein the silver particles comprise both silver in thezero-valent, that is metallic, oxidation state [Ag(0)] and a coating ofsilver oxide with the stoichiometry AgO.

Experimental evidence shows that AgO in the particles of the presentinvention is at least partially in the form of Ag₄O₄—that is, silver IIoxide. In a molecule of this material two of the silver atoms are in the1⁺ state (silver I) while the other two silver molecules are in the 3⁺state (silver III). Under certain conditions these molecules can giverise to silver atoms in the 2⁺ (silver II) state.

A further class of embodiments is the combination of any of theabove-described embodiments with hydrogen peroxide, at a level of 1-3wgt % hydrogen peroxide in the final product.

EXAMPLES Formation of Composition

Compositions of silver in water can be made according to procedures setforth in U.S. Pat. No. 6,214,299, incorporated by reference herewith.

A preferred method for producing a composition comprising silveraccording to this invention utilizes a electrochemical cell comprisingelectrodes and comprises the steps

(a) placing a silver electrode in contact with a quantity of high puritywater;

(b) conveying electrical current through the silver electrode to therebyseparate particles of silver from said silver electrode in a mannersufficient to cause production of suspended silver particles within thewater; and

(c) agitating the water during said production of suspended silverparticles to thereby disperse the silver particles into a more uniformconcentration within said water such that a higher quantity of suspendedsilver particles can be produced per batch.

Another preferred method for producing a composition comprising silverutilizes an electrochemical cell and comprises the steps of:

(a) establishing an electrical circuit comprising a current source, anda first conductor electrically connected to said current source and asecond conductor electrically connected to said current source, whereinsaid first conductor is disposed spaced apart from said secondconductor, and wherein at least one of the conductors is made ofelemental silver;

(b) closing the circuit by placing the first conductor and the secondconductor in communication with a fluidic resistor;

(c) operating the current source to supply alternating currentsimultaneously to the first conductor and the second conductor such thatvoltage is increasing and decreasing within the first and secondconductors in alternating tandem to thereby cause silver particles toseparate from the first electrode and enter the fluidic resistor andbecome disposed in suspension within said fluidic resistor; and

(d) selectively adjusting the electrodes by moving them toward thefluidic resistor to compensate for decrease in electrode length due togradual separation of silver particles therefrom to thereby preventarcing from occurring between the electrodes and said fluidic resistor.

The analysis of the silver content in the silver compositions of thisinvention may be done by atomic absorption (AA), inductively coupledplasma/atomic emission (ICP/AES), or other techniques known to one ofordinary skill in the art to be sensitive to silver in the appropriateconcentration range. If the particles of the silver composition aresmall and uniformly sized (for example, 0.01 micrometers or less), areasonably accurate assay may be obtained by running the colloiddirectly by AA or ICP/AES. This is because the sample preparation for AAionizes essentially all of the silver allowing its ready detection.

If the compositions comprise particles as large as 0.2 micrometers, itis preferred to use a digestion procedure. The digestion procedure isnot necessarily ideal for silver compositions that may have beenmanufactured or stored in contact with halides or other anionic speciesthat may react with finely divided silver, or combined with protein orother gelatinous material. An embodiment of the digestion procedure isas follows:

1 Take a 10 ml aliquot of a thoroughly mixed or shaken silvercomposition to be analyzed, and place it in a clean polycarbonate bottleor other container of suitable material (generally, the bottle) with atight fitting lid. A size of 30-100 ml is preferred.

2 With a micropipette or dropper, add 0.1 ml of nitric acid, reagentgrade to the silver composition in the bottle.

3 With the lid of the bottle tightly in place, heat the silvercomposition to 80° C. with mild agitation for a time sufficient todissolve the silver—dissolution is essentially instantaneous.

4 Allow the resulting mixture to cool to room temperature with the lidin place. Shake the bottle thoroughly.

5 Utilize AA, ICP/AES, or equivalent means to analyze the silver contentof the silver mixture. Preferably, one will utilize a freshly preparedstandard or standards, preferably prepared according the equipmentmanufacturer's instructions, with appropriate dilution as needed.

6 When reporting results, one must taken into account all dilutionsduring preparation, including the 1% dilution caused by addition of thenitric acid.

Analysis of Physical/Chemical Form of Silver

A. Introduction

A sample of a composition, nominally containing 22 ppm silver in water,was analyzed by time-of-flight secondary ion mass spectrometry(TOF-SIMS) in order to determine the form of silver in the composition.The conclusion is that the bulk of the silver exists as silver (0) [thatis, metallic silver] and that there is a surface coating which as onaverage a composition of silver (II) oxide [AgO]. As mentioned abovesilver (II) oxide is usually a stoichiometric combination of silver (I)and silver (III).

B. Experimental Procedure

A few drops of the 22 ppm inventive silver composition were evaporatedto dryness on a silicon substrate at ambient temperature. The residuewas analyzed by TOF-SIMS, and is denoted as the sample. A referencesilver (II) oxide (AgO) material was analyzed by placing a few particlesof the reference powder as received from the vendor on a siliconsubstrate, and is denoted as the reference.

The Time-of-Flight Secondary Ion Mass Spectrometry technique (TOF-SIMS)is based on the principle of bombarding a solid sample with a pulsed,finely focused beam of primary ions, and then analyzing the secondaryions produced from the surface of the sample via a time-of-flight massspectrograph. This analytical technique is surface sensitive, derivingits information from a layer that extends to approximately 20 to 40 Å(one Angstrom=1×10−4 micrometers) below the surface. The TOF-SIMStechnique is normally used as a survey tool to identify the compositionof unknown samples. It is capable of quantification if the appropriatemicroanalytical standards are available for calibration. This analysiswas carried out using standard high mass-resolution conditions.

C. Results

Negative ion mass were obtained for the Ag(II)O reference material andthe product sample, respectively. The mass spectral region for bothspectra showed the presence of AgO— species. The data suggest thatsilver (II) is the average oxidation state of the silver present on thesurface of the sample particles. The silver oxide (AgO) signals exhibitsignificantly higher intensity in the reference sample compared to theproduct sample which is probably because metallic silver is dominant inthe sample. It will be appreciated that as the average particle size inthe sample is decreased the ratio of silver to silver oxide will alsodecrease as more silver oxide will be present.

Size Analysis

It is likely that the unusual effectiveness of the silver preparationsdescribed herein is due to the relationship between the surfaceproperties/inner properties (i.e., oxide/metal) of the particles and thesize distribution of the particles. The smaller the average particlesize, the greater the surface area and the greater the contribution ofthe particular surface chemistry. However, if the particles areexcessively small there can be a loss of stability and/or otherinteractions that negatively affect the product. The silver compositionsof the instant invention are remarkable because they are stable inessentially pure water without surfactants, etc. Also, the materials areessentially colorless while other colloidal silver preparations(particularly with larger particle sizes) usually show colors. Theseproperties are a result of the exact manufacturing conditions asdiscussed above.

Digital analysis of the composition showed that there is an averageparticle diameter of 0.0106 micrometers with a range of 0.005 micrometerto 0.0851 micrometers. However, size distribution analysis shows thatmore than 95% of the particles were between about 0.005 micrometers andabout 0.015 micrometers in diameter.

Evidence of Efficacy of 22 ppm Silver Composition Against BacillusSubtilis

A. Purpose of Example

The purpose of this example is to demonstrate the antimicrobial activityof the silver-based composition of the present invention on bacterialendospores from the test organism Bacillus subtilis. This wasaccomplished by performing a standard kill-time assay using a suspensionof B. subtilis endospores. Normally, bacterial endospores are resistantto killing.

B. Material and Methods

Test Organism.

A test suspension containing endospores from Bacillus subtilis (ATTC#19659) was prepared from a culture grown on nutrient agar, to whichadditional sporulation enhancement ingredients were added. Plates wereharvested with sterile water and endospores were purified by repeatedcentrifugations and resuspensions in water. The final wash was in 70%ethanol for 30 min, to ensure the destruction of all vegetativebacteria. The spores were resuspended in water containing 0.1% Tween 80(brand of polysorbate surfactant) to prevent clumping.

Neutralizer.

The Neutralizer mixture consisted of 12.7% Tween® 80 (brand ofpolysorbate), 6.0% Tamol® SN (brand of sodium salt ofnaphthalene-formaldehyde condensate), 1.7% lecithin, 1% Peptone, and0.1% Cystine. This solution was intended to neutralize any chemicals sothey would not affect subsequent growth of the bacteria.

Kill-Time Procedure:

a) A 9.9 ml aliquot of the disinfectant (inventive 22 ppm silvercomposition, in water) was placed in a sterile 20 mm×150 mm tube. Thetube was equilibrated in a 20° C. water bath.

b) A 9.9 ml aliquot of the disinfectant (inventive 22 ppm silvercomposition, in water) was placed in a sterile 20 mm×150 mm tube. Thetube was equilibrated in a 20° C. water bath.

c) At 30 min. 1 hr, and 4 hr, one ml of organism/disinfectant suspensionwas removed to a tube containing nine ml of Neutralizer. The tube wasmixed thoroughly.

d) After two min, the neutralized suspension was serially diluted 1:10,in physiological saline solution (PSS).

e) The number of viable organisms in selected dilution tubes was assayedby membrane filtration. One ml aliquots were plated in duplicate. Themembranes were washed with about 100 ml of sterile PSS and removed toNutrient Agar plates. The plates were incubated at 37° C. for 20 hr.

f) The number of colonies on each filter was counted and log reductionswere computed.

Controls:

a) Titers of the test suspensions were computed by performing membranefiltration assays of selected 1:10 dilutions of the test suspensions inPSS.

b) A neutralizer control was performed by inoculating a mixture of 9 mlneutralizer and 1 ml of disinfectant with 100 μl of a dilution of thetiter containing 100 cfu. This produced about 10 cfu/ml in the tube,which was allowed to stand for 20 minutes prior to assay by membranefiltration using duplicate 1 ml samples.

C. Results

Bacillus subtilis Titer:

Dilution: 1:1 × 10⁶ 1:1 × 10⁷ 1:1 × 10⁸ Number of colonies: TNTC 75 7TNTC 58 8 TNTC = too numerous to count

Dilution of B. subtilus Spore/Disinfectant Suspension:

1:1 × 1:1 × Time 1:1 × 10¹ 1:1 × 10² 1:1 × 10³ 1:1 × 10⁴ 10⁵ 10⁶ 30 — —TNTC TNTC 57 10 min — — TNTC TNTC 51 7 1 hr — — TNTC TNTC 28 3 — — TNTCTNTC 55 3 2 hr — TNTC TNTC 126 23 — — TNTC TNTC 183 17 — 4 hr TNTC TNTC88 12 — — TNTC TNTC 69 12 — — TNTC = too numerous to countNeutralization Control: 1:1 × 10⁸

D. Discussion

Results of the titer showed a viable B. subtilis spore concentration of6.65×10⁸ spores per ml in the original suspension. Inoculation of 9.9 mlof disinfectant with 100 μl of this suspension produced an initialconcentration of 6.65×10⁶ spores per ml in the assay tube.

Results from these procedures allowed log reductions (LR) and PercentKill (PK) values to be calculated. They are listed in the table below.Values were computed using the formulae: LR=−Log(S/So) andPK=(1−(S/So))×100; where S=concentration of organisms at a specifictime; and So=the initial concentration of organisms at time zero.

Time LOG REDUCTION PERCENT KILL 30 min 0.090 18.8  1 hr 0.205 37.6  2 hr0.634 76.8  4 hr 1.928 98.8

Neutralization control data showed that the disinfectant was adequatelyneutralized. Actual counts correspond to those resulting from dilutionwithout appreciable killing.

The disinfectant preparation tested here displayed good sporicidalactivity against B. subtilis spores. B. subtilis is a common speciesused in sporicidal testing and belongs to the same genus as the organismthat causes anthrax. Because of their genetic similarities, B. subtilisspores have been used as a non-pathogenic surrogate for Bacillusanthracis, the anthrax bacterium. Therefore, these results areapplicable to anthrax. It is expected that longer exposure would resultin additional killing.

Evidence of Efficacy of 10 ppm Silver and 1.0% H₂O₂ Composition and 14ppm Silver and 1.5% H₂O₂ Composition Against Bacillus Subtilis

A. Purpose of Example

The purpose of this example is to demonstrate the antimicrobial activityof two silver-based compositions of the present invention on bacterialendospores from the test organism Bacillus subtilis. This wasaccomplished by performing standard kill-time assays using a suspensionof B. subtilis endospores. Viewed relative to the previous example(employing 22 ppm silver), this example establishes the promoting effectof hydrogen peroxide (H₂O₂) on the antimicrobial properties of silvercompositions. Hydrogen peroxide is stable in the presence of the silvercompositions of the present invention. While hydrogen peroxide hassignificant antimicrobial properties itself, it is frequently brokendown by catalase or other microbial enzymes. However, the hydrogenperoxide is capable of weakening bacterial cell walls and enhancingentry of the silver particles before any enzymatic destruction of thehydrogen peroxide can occur.

B. Material and Methods

1 Test Organism.

A test suspension containing endospores from Bacillus subtilis (ATCC#19659) was prepared from a culture grown on Nutrient Agar, to whichadditional sporulation enhancers were added. Plates were harvested withsterile water and endospores were purified by repeated centrifugationsand resuspensions in water. The final wash was in 70% ethanol for 30min, to ensure the death of all vegetative bacteria. The spores wereresuspended in water containing 0.1% Tween® 80 (brand of polysorbate) toprevent clumping.

2 Neutralizer.

The Neutralizer mixture consisted of 12.7% Tween 80, 6.0% Tamol® SN(brand of sodium salt of naphthalene-formaldehyde condensate), 1.7%lecithin, 1% Peptone, and 0.1% Cystine. This solution was intended toneutralize any chemicals so they would not affect subsequent growth ofthe bacteria.

3 Kill-time Procedure:

a) A 9.9 ml aliquot of each of the disinfectants (inventive colloidalsilver compositions: one containing 14 ppm silver and 1.5% H202; theother containing 10 ppm silver and 1.0% H₂O₂) was placed in a sterile 20mm×150 mm tube. The tubes were equilibrated in a 20° C. water bath.

b) Each tube of disinfectant was inoculated with 100 μl of the testorganism suspension at time zero.

c) At 10 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr, and 8 hr, one ml oforganism/disinfectant suspension was removed to a tube containing nineml of neutralizer. The tube was mixed thoroughly.

d) After two min, the neutralized suspension was serially diluted 1:10,in physiological saline solution (PSS).

e) The number of viable organisms in selected dilution tubes was assayedby membrane filtration. One ml aliquots were plated in duplicate. Themembranes were washed with about 100 ml of sterile PSS and removed toColumbia Agar plates. The plates were incubated at 37° C. for 20 hr.

f) The number of colonies on each filter was counted and log reductionscomputed.

4. Controls:

a) Titers of the test suspensions were computed by performing membranefiltration assays of selected 1:10 dilutions of the test suspensions inPSS.

b) A neutralizer control was performed by inoculating a mixture of 9 mlof neutralizer and 1 ml of disinfectant with 100 μl of the 1:10³dilution of the titer. This produced about 2,000 cfu/ml in the tube,which was allowed to stand for 20 minutes prior to diluting 1:10. Bothtubes were assayed by membrane filtration using duplicate 1 ml. samples.

C. Results

Titer of Bacillus subtilis Spores:

Dilution: 1:1 × 10⁶ 1:1 × 10⁷ 1:1 × 10⁸ Number of colonies: TNTC 36 5TNTC 27 4 TNTC = too numerous to count.

Solution containing 14 ppm silver and 1.5% H₂O₂:

Dilution of B. subtilis Spore/Disinfectant Suspension:

Time 1:1 × 10¹ 1:1 × 10² 1:1 × 10³ 1:1 × 10⁴ 1:1 × 10⁵ 10 min — — TNTCTNTC 227 — — TNTC TNTC 265 30 min — — TNTC TNTC 258 — — TNTC TNTC 273  1hr — — TNTC TNTC 55 — — TNTC TNTC 33  2 hr — TNTC 207 29 — — TNTC 237 24—  4 hr 59 3 1 57 5 1  6 hr 0 0 0 3 0 0  8 hr 1 0 0 1 0 0 TNTC = toonumerous to count.

Neutralization Control:

Undiluted 1:1 × 10¹ TNTC 195 TNTC 210 TNTC = too numerous to count.

Solution containing 10 ppm silver and 1.0% H₂O₂:

Dilution of B. subtilis Spore/Disinfectant Suspension:

Time 1:1 × 10¹ 1:1 × 10² 1:1 × 10³ 1:1 × 10⁴ 1:1 × 10⁵ 10 min — — TNTCTNTC 230 — — TNTC TNTC 287 30 min — — TNTC TNTC 254 — — TNTC TNTC 260  1hr — — TNTC TNTC 146 — — TNTC TNTC 124  2 hr — TNTC TNTC 64 — — TNTCTNTC 71 —  4 hr TNTC 72 5 TNTC 77 5  6 hr 0 0 0 2 0 0  8 hr 0 0 0 0 0 0TNTC = too numerous to count.

Neutralization Control:

Undiluted 1:1 × 10¹ TNTC 200 TNTC 184 TNTC = too numerous to count.

D. Discussion

The data showed a viable B. subtilis spore concentration of 2.59×10⁸spores per ml in the original suspension. Inoculation of 9.9 ml ofdisinfectant with 100 μl of this suspension produced an initialconcentration of 2.59×10⁵ spores per ml in the assay tube.

Results from these procedures allowed log reductions (LR) and PercentKill (PK) values to be calculated. They are listed in the followingtable. Values were computed using the formulae: LR=−Log(S/So) andPK=(1−(S/So))×100; where. S=concentration of organisms at a specifictime; and So=the initial concentration of organisms at time zero. Sincethere was no significant kill within 30 min, the 10 min data was usedfor the So values. The 6 hr and 8 hr exposure times did not producecounts high enough to be reliable. Therefore, these data were not usedin the linear regressions. Linear regressions were performed on the logreduction values using the ‘fitted line plots’ command in the Minitabstatistical software package. The regression equations produced, and thetimes required to effect a six-log reduction are shown along with thelog reduction and percent kill values in the following table.

14 ppm SILVER + 10 ppm SILVER + 1.5% H₂O₂ 1.0% H₂O₂ LOG PERCENT LOGPERCENT Time REDUCTION KILL REDUCTION KILL 30 min −0.03 −7.9 0.003 0.6 1 hr 0.66 78.0 0.28 47.8  2 hr 2.05 99.1 1.58 97.4  4 hr 4.63 99.9983.54 99.97

Regression Analysis

Equation for 14 ppm calculated line: Y=−0.66704+1.32936x. Equation for10 ppm calculated line: Y=−0.59690+1.03933x. These equations predictthat the time for a 6-log reduction is 5.02 hrs for the 14 ppmcomposition and 6.35 hrs for the 10 ppm composition.

The neutralization control data showed that the disinfectant wasadequately neutralized. Expected counts corresponded to those expectedfrom the dilution.

The experimental disinfectant solutions tested exhibited significantsporicidal activity against B. subtilis spores. The B. subtilis strainused in these evaluations is the same one specified in the AOACsporicidal test. Spores from this organism represent a significantchallenge for most disinfectants. The times required to effect a six logreduction are in line with the sporicidal label claims of many coldsterilants.

Evidence of Efficacy of 10 ppm Silver Composition as a Broad SpectrumAntimicrobial

A. Methods

MIC (minimum inhibitory concentration) and MBC (minimum bactericidalconcentration) tests were performed according to the standard brothmicrodilution method. The MIC is defined as the lowest concentration ofan antibiotic that will inhibit the (in vitro) growth of an infectiousorganism. Results are reported in micrograms per ml. For medicalantibiotics the interpretation of in vitro data is based on achievableserum concentrations of the drug, which may vary depending on dose,route of administration, degree of protein binding, site of infection,age and weight of the patient, and other factors. The MBC is defined asthe lowest concentration of an antimicrobial agent needed to kill 99.9%of the initial organism inoculum.

The test was preformed by growing pure cultures of each of the testorganisms in liquid culture. Turbidometric measurements were used tocontrol the concentration of the culture. Serial dilutions of each testantibiotic were made in nutrient broth. The dilutions were calculated tocover the susceptible ranges for each organism for each agent. Astandard amount of the test culture was added to each tube and the tubereturned to an incubator (37±2° C.) for growth. The tubes were checkedturbidometrically to determine bacterial growth. Below the MICconcentration the tubes showed an increase in optical density with timeindicating bacterial growth. The lowest concentration of the antibioticthat showed no growth was the MIC. The “no growth” tubes were thensubcultured in fresh medium. The “no growth” tube with the lowestconcentration of antibiotic that showed no growth on subculturing wasthe MBC.

B. Results:

Antimicrobial (ppm) Organism Tetracycline Ofloxacin Penicillin GCefaperazone Erythromycin Silver S. pyogenes 0.625/>5 1.25/2.5  >5.00.313/1.25  0.003/0.019 2.5/5.0 S. mutans 0.625/>5  2.5/>5.0 0.521/>5  1.25/>5   0.009/0.019  2.5/10.0 S gordonii  0.156/0.625 2.5/5.00.009/0.039 1.25/1.25 0.005/0.019  2:5/10.0 S. pneumoniae  0.078/0.6252.5/2.5 0.019/0.019 0.313/0.313 0.002/0.004 2.5/2.5 S. faecalis 0.313/>51.25/5.0   5.0/>5.0 >5.0 0.009/1.25  10.0/10.0 S. aureus 0.313/>50.417/0.625  2.5/>5.0 5.0/5.0 0.039/>5.0  5.0/5.0 P. aeruginosa 0.078/5 0.156/0.313 0.13/>5.0 2.5/5.0  2.5/>5.0 1.67/5   E. coli  1.67/>50.104/0.156 >5.0 0.625/>5.0   5.0/>5.0 2.5/2.5 E. aerogenes >50.078/0.156 >5.0 2.92/>5.0 >5.0 2.5/2.5 E. cloacae  1.67/>50.156/0.156 >5.0 >5.0 >5.0 2.5/5.0 S. typhimurium  1.25/>50.078/0.156 >5.0 1.25/2.5   5.0/>5.0 2.5/5.0 S arizona 0.625/>50.078/0.078 >5.0 0.833/>5.0  4.17/>5.0 2.5/5.0 S. boydii  1.25/>50.078/0.156 >5.0 0.625/0.625  5.0/>5.0 1.25/1.25 K. pneumoniae  2.5/>50.417/0.625 >5.0 >5.0 >5.0 2.5/2.5 K. oxytoca  1.25/>510.104/0.156  >5.0 1.25/>5.0 >5.0 1.25/1.25

Data are presented as MIC/MBC (minimum inhibitory concentration/minimumbactericidal concentration) in parts per million (ppm)); “>” denotesthat the concentration needed to obtain the MIC or the MBC was higherthan test parameters measured for the test. For example, the highestconcentration of tetracycline used on S. pyogene was 5 ppm. At thatconcentration there was still growth upon subculturing of the “nogrowth” tubes. Therefore, the MBC must be >(greater than) 5 ppm.

The MIC/MBC of E. coli strain O 157:H7, which has been associated withoutbreaks of hemorrhagic diarrhea and colitis, was determined in asubsequent study. The MIC was determined to be 2.5 ppm and the MBC wasdetermined to be 5 ppm.

C. Conclusion

The 10 ppm silver composition of the present invention was tested andfound to be both bacteriostatic and bactericidal for all organismstested. In other studies, this composition was compared to othercommercially available colloidal silver products and found to have asuperior activity to all other preparations tested (data not shown). Themost interesting observation was the broad spectrum that the 10 ppmsilver composition possesses. The antimicrobial activity that wasobserved was fairly constant independent of the particular organismtested. With the exception of Streptococcus faecalis and Streptococcusaureus (which had MIC values of 10 ppm and 5 ppm, respectively), MICvalues ranged between 1.25 ppm and 2.5 ppm for both gram positive andgram negative organisms. The MBC values behaved similarly with valuesranging from 1.25 ppm to 5 ppm with the exception of Streptococcusmutans, Streptococcus gordonii, and Streptococcus faecalis (which allhad MBC values of 10 ppm). The data suggest that 10 ppm silverembodiment of this invention exhibits an equal or broader spectrum ofactivity than any one antibiotic tested. Antibiotics generally haverestricted antibacterial spectra limited to susceptible organisms, butas the data demonstrate, the silver composition of the present inventionis equally effective against both gram positive and gram negativeorganisms. The data suggest that with the low toxicity associated withsilver, in general, and the broad spectrum of antimicrobial activity ofthis silver composition, this preparation can be effectively used as analternative to antibiotics.

D. Reference for Preceding Example

1 U.S. EPA IRIS Report for Silver-CASRN 7440-22-4

2 Fox C L, Modak S M. Mechanism of Silver Sulphadiazine Action on BurnWound. Infections. Antimicrobial Agents Chemother. 5:582-588.1974.

3. Furchner, J E, Richmond C R, and G A Drake. Comparative Metabolism ofRadionuclides in Mammals. IV. Retention of Silver-110m in the Mouse,Rat, Monkey, and Dog. Health Phys. 15:505-514.1968.

4. Grier, N. Silver and its Compounds in Disinfection, Sterilization,and Preservation. (Seymour S. Block, ed) 2^(nd) Edn, pp 395-407. 1977.

5. Hindler, J A, and J H Jorgensen. Procedure in Antimicrobial Testingin Diagnostic Microbiology. (CR Mahon and G Manuselis, eds) pp63-91.1995.

Evidence of Efficacy of 32 ppm Silver Composition Against PseudomonasAeruginosa, Salmonella Choleraesuis and Staphylococcus Aureus

A. Methods

Pseudomonas aeruginosa ATTCC #15442, Salmonella choleraesuis ATTCC#10708 and Staphylococcus aureus ATCC #6538 were tested using the AOAC(Association of Official Analytical Chemists AOC Methods, vol. 1,15^(th) edition, 1990, AOAC Arlington, Va.) official methods 955.14,95515 and 964.02. Nutrient broth (NBAOAC) tubes were inoculated from thestock culture, and the tubes incubated at 37±2° C. Transfers to freshtubes of nutrient broth were made for three successive days with thefinal transfer being incubated at 37±2° C. for 48-54 hr. The Pseudomonasculture was decanted into a fresh tube to remove the pellicle. The othercultures were vortexed for 3-4 seconds and allowed to stand for 10 minat room temperature. Finally the cultures were diluted 1:100 in peptonewater (PEPW) to which equine serum was added to yield a 5% total organicchallenge. Test carriers (10 mm long polished 304 stainless steelcylinders with an 8 mm outside diameter and 6 mm inside diameter) weresoaked in challenge solution for 15 min, removed, drained and dried at37±2° C. for 40±2 min prior to use.

Phenol Resistance.

Five-one ml aliquots of each dilution of the test phenol were placedinto sterile test tubes and allowed to equilibrate in a 20±2° C. waterbath. At 30 second intervals, 0.5 ml of each challenge culture was addedto the appropriate dilutions of phenol, agitated, and replaced into thewater bath: After the appropriate exposure times of 5, 10, and 15minutes, a loopful of suspension was removed from the assay tubes andtransferred to tubes of letheen broth (LETH). The tubes of LETH wereincubated at 37±2° C. for 2 days.

Carrier Titration.

For titration of carriers, 10 ml blanks of peptone Tween® (brand ofpolysorbate) (PEPT) solution were prepared. Two carriers were placedinto the individual tubes, representing the first 1:10 dilution. Thetubes were agitated vigorously enough to get bacteria into solution andserial dilutions were made into 9 ml blanks of LETH medium. The dilutionblanks were incubated at 37±2° C. The last tube with growth indicatedthe logo titer of organisms on the carrier. AOAC requires carriers tohave minimum populations of 1×10⁴ cfu/carrier.

Test of Silver Composition.

Using sterile glass pipettes, 10 ml aliquots of the prepareddisinfectants were placed into sterile test tubes and allowed toequilibrate in a refrigerated water bath held at 20±2° C. Withouttouching the sides of the test tubes, one contaminated dried carrier wasadded at 30 second intervals to each tube of silver composition andplaced back into the water bath. For each organism the disinfectant wastested against 60 dried contaminated carriers at 5 and 10 minuteexposure intervals. Following exposure, the carriers were removed fromthe disinfectant and transferred to a tube of LETH. The culture tubeswere incubated at 37±2° C. for 2 days and scored as positive (+) ornegative (0) for growth of the challenge organism.

Controls.

For each organism, a dried contaminated carrier was added to a tube ofLETH as a positive control. Uninoculated media tubes served as negativecontrols. After incubation, all negative tubes were spiked with 1-100colony forming units (cfu) of the corresponding organisms to demonstrateneutralization efficacy. To demonstrate growth promotion of the media,the negative control tubes were also inoculated with the same 1-100 cfufor all three organisms. The inoculating volumes were plated intriplicate onto soybean casein digest agar (SCDA) to verify theinoculating titers. The tubes and plates were incubated at 37±2° C.until growth was seen in all tubes.

On the P. aeruginosa neutralization, the initial titer of inoculum wasfound to be >100 cfu which was too high for the protocol. Because alloriginal tubes had been spiked, a simulated test was performed with samelot of media used in testing by placing carriers into disinfectant tubesfrom all three lots of silver compositions for 10 minutes. The carrierswere sub-transferred to LETH blanks. These tubes were then spiked with1-100 cfu of organism. The tubes were incubated as before and scored forgrowth or no growth. New tubes of sterile media from the same lot werealso inoculated as a growth promotion verification.

B. Results

Initial testing using S. aureus demonstrated passing results for sample#1 and #2, but sample #3 failed. Upon investigation it was decided thatsample #3 may have been damaged prior to shipment. A new bottle wasobtained from the same lot as sample #3, and the new bottle was labeledas sample #4. The S. aureus challenge was repeated using sample #4. AOACguidelines state that for any one time point and organism, only 1carrier is allowed for growth for each lot tested.

Positive controls demonstrated growth and negative controls demonstratedno growth for all lots, time points, and organisms.

Carrier titration was run in duplicate for all organisms. The reportedtiter is an average of the replicates. For all three organisms, theaverage titer found on the carriers ranged from 5.5×10⁴ to 5.5×10⁶cfu/carrier. AOAC requires carriers to have a minimum of 1.0×10⁴cfu/carrier.

For P. aeruginosa 3/180 carriers showed growth at the 5 min test pointand 2/180 carriers showed growth at the 10 min test point. For S. aureus16/180 carriers showed growth at the 5 min test point and 2/180 carriersshowed growth at the 10 min test point. For S. choleraesuis 6/180carriers showed growth at the 5 min test point and 1/180 carriersshowed-growth at the 10 min test point.

The test Pseudomonas culture showed growth following a 5, 10 or 15 mintreatment with 1:90 phenol and showed growth following a 5 or 10 mintreatment with 1:80 phenol but no growth following 15 min treatment with1:80 phenol. The Staphylococcus culture showed growth following a 5, 10or 15 min treatment with 1:70 phenol and showed growth following 5 or 10min treatment with 1:60 phenol but no growth following a 15 mintreatment with 1:60 phenol. The Salmonella culture showed growthfollowing a 5, 10 or 15 min treatment with 1:100 phenol but no growthfollowing a 5, 10 or 15 min treatment with 1:90 phenol.

Evidence of Effectiveness of 32, 22, and 10 ppm Silver and 22 ppm Silverand 1.5% H₂O₂ and 10 ppm Silver and 10 ppm K₂S₂O₈ Against Salmonella andEscherichia coli in Freshly Inoculated Beef Samples

A. Purpose of Example

The purpose of this example is to demonstrate the antimicrobial activityof the silver-based composition embodiments of the present invention onsamples of beef flank steak inoculated on the exterior surface with afive strain cocktail of Salmonella species. or Escherichia coli O157:h7at a high inoculum solution level (1×10⁶ cfu/cm²) and separately at alow inoculum solution level (1×10⁴ cfu/cm²) (cfu=colony forming unit).

B. Material and Methods

Beef Samples.

Beef tissue samples were obtained from slaughter houses within 8 hoursof evisceration. The rectus abdominus muscle was peeled off carcasseshanging in the chill cooler by making an incision between the 11^(th)and 12^(th) ribs and then peeling the muscle out along the natural seam.The aseptically retrieved samples were placed in plastic bags and on icepacks and were transported on the same day to the laboratory, where thesamples were promptly packed in a Multi-Vac (A-300) and placed in a 4°C. cooler. Samples used for testing had a pH between 5.8 and 6.0 andwere no more than 36 hours post evisceration. From randomly selectedrectus abdominus muscles, 13×8 cm samples were cut and treated. Aftertreatment, a 3.5 cm² flame sterilized stainless steel coring device andsurgical scalpel were utilized to aseptically retrieve two meat coresper sampling interval from each sample. Tissue cores were placed in asterile stomacher bag with 25 ml of 0.1% peptone and were mixed for twominutes in a stomacher (Lab Bender 400). Serial dilutions were preparedand spiral plated at 0 minutes, 20 minutes, 1 hour, 4 hours, and 24hours post-treatment on selective and recovery media.

Bacterial Cultures.

Bacterial cultures were obtained from the Kansas State University (KSU)stock culture collection and were stored using the “Protected Bead”storage system. The following cultures were used for the Salmonellaspecimen: S. lille (UGA), S. montevideo (UGA), S. typhimurium (UGA), S.agona (KSU 05 from CDC outbreak isolate), and S. newport (KSU 06 CDCoutbreak isolate). The following cultures were used for the Escherichiacoli specimen: E. coli O157:H7 (CDC 01,03), E. coli O157:H7 (USDA-FSIS011-82 Rif resistant 100 ppm), E. coli 157:H7 (ATCC 43895 HUS associatedType I & II toxins Rif. Res.) and E. coli ATCC#23740 (Genotype K-12prototrophic lambda).

Stock cultures were cultivated by placing one impregnated bead into a 5ml solution of Difco® Tryptic Soy Broth (TSB) and incubating for 24hours at 35° C. Next, a 0.05 ml loop of the respective culture wasinoculated into a 5 ml solution of TSB and incubated for 24 hour at 35°C. to obtain a pure culture. After incubation, 1 ml of the respectiveculture was inoculated into 49 ml TSB and incubated for 24 hours at 35°C. Following incubation, samples were centrifuged (15,300×g at 4° C.),and the supernatant material decanted and the pellet was re-suspendedwith 50 ml of 0.1% peptone and centrifuged (15,300×g at 4° C.) a finaltime. The peptone was decanted and the remaining pellet was re-suspendedwith 10 ml of 0.1% peptone. The five 10 ml bottles of respective culturewere mixed together to create a 50 ml cocktail containing 10⁹ cfu/ml ofSalmonella species. The cocktail was diluted to 10⁶ cfu/ml or 10⁴ cfu/mlusing 0.1% peptone. Cultures were confirmed by cultivation on selectiveand differential media, and biochemical analysis of presumptive coloniesusing API 20E kits.

Method of Inoculation.

Samples of beef flank steak (rectus abdominus muscle) were trimmed to13×8 cm (104 cm²) and were inoculated with a five strain cocktail ofSalmonella species. or Escherichia coli O157:h7 at a high inoculumsolution level (10⁶ log cfu/cm²) and separately at a low inoculumsolution level (10⁴ log cfu/cm²). This inoculum was misted onto thetissue surface using a plastic spray bottle with samples containedwithin a sealed inoculum chamber. The actual Salmonella species.concentration on the meat surface was approximately 5.0 and 3.4 logcfu/cm² for the high and low level inoculum solution, respectively. ForE. coli O157:H7, the respective meat surface inoculation levels were 4.2and 3.9 log cfu/cm².

The beef samples were then hung vertically on stainless steel hooksattached to a motorized track that pulled the beef samples through amodel spray cabinet (Kansas State University, Food Safety Laboratory)while spray treatments were applied. Treatments with either the silvercompositions of this invention or deionized water were applied to thebeef at 20 psi from a distance of 13 cm in the model pressure rinsecabinet for 20 seconds. The spray nozzle (BETE NF0580 303) deliveredapproximately 20 ml of solution to the surface of the beef sample. Thetemperature of solutions and treatment application room wasapproximately 14° C. After treatment, duplicate 3.5 cm² core sampleswere randomly drawn from the lateral surface of the beef sample at 0,20, 60 and 240 minutes. Samples were cultivated and enumerated onselective differential and recovery media. Log reductions werecalculated by subtracting the log₁₀ of cfu/cm² of the inoculated/treatedsamples at the specified sampling times (0, 20, 60, and 240 minutes)from the log₁₀ of cfu/cm² of the inoculated/untreated samples at 0minutes. Sample treatment included the use of 32 ppm silver, 22 ppmsilver, and 10 ppm silver compositions according to the presentinvention. Separately, combinations of 22 ppm Ag with 1.5 wght %hydrogen peroxide and 10 ppm Ag with 10 ppm peroxydisulfate (K₂S₂O₈)were tested.

C. Results with 32 ppm Silver Composition

The use of a composition of 32 ppm silver according to this inventionproduced a reduction in bacteria on beef steak. In the following, thisreduction is expressed as the log₁₀ of the ratio of the number ofbacteria in the control at time 0 to the amount of bacteria in thetreated specimen at the sampling (i.e., treatment) time.

For Salmonella, at the lower initial bacteria level (10⁴), the followinglog reductions were recorded: 0.78 at 0 minutes, 1.11 at 20 minutes,1.08 at 60 minutes, and 1.23 at 240 minutes. Thus, at 4 hours (240minutes), the ratio of the initial bacteria count in the control tobacteria in the sample treated with 32 ppm silver is 10^(1.23). For thehigher initial bacteria level (10⁶), the following log reductions wererecorded: 0.86 at 0 minutes, 0.95 at 20 min, 0.98 at 60 min and 1.17 at240 min. The results indicate that the 32 ppm silver embodiment of thisinvention shows an effective bactericidal effect for Salmonella on beefsteak. It will be appreciated that disinfecting a meat surface is anextreme challenge for any disinfectant.

For E. coli, for the lower initial bacteria level (10⁴), the followinglog reductions were recorded: 1.03 at 0 minutes, 1.28 at 20 minutes,1.42 at 60 minutes, and 1.58 at 240 minutes. For the higher initialbacteria level (10⁶), the following log reductions were recorded: 0.65at 0 minutes, 0.60 at 20 minutes, 0.83 at 60 minutes and 0.87 at 240minutes. The results indicate that the 32 ppm silver embodiment of thisinvention shows an effective bactericidal effect for pathogenic E. colion beef steak.

D. Results with 22 ppm Silver Composition

Results with Silver in Water.

For Salmonella at the lower initial bacteria level (10⁴), the followinglog reductions were recorded: 0.41 at 0 minutes, 0.43 at 20 minutes,0.48 at 60 minutes, and 0.68 at 240 minutes. For the higher initialbacteria level (10⁶), the following log reductions were recorded: 0.24at 0 minutes, 0.24 at 20 minutes, 0.42 at 60 minutes and 0.61 at 240minutes. The results indicate that the 22 ppm silver embodiment of thisinvention furnishes an effective bactericidal effect for Salmonella onbeef steak.

Results with Silver in Water and 1.5 wght % Hydrogen Peroxide.

For Salmonella, for the lower initial bacteria level (10⁴), thefollowing log reductions were recorded: 0.34 at 0 minutes, 0.33 at 20minutes, 0.36 at 60 minutes, and 0.62 at 240 minutes. For the higherinitial bacteria level (10⁶), the following log reductions wererecorded: 0.28 at 0 minutes, 0.14 at 20 minutes, 0.30 at 60 minutes and0.69 at 240 minutes. The results indicate that the 22 ppm silver with1.5 wght % hydrogen peroxide embodiment of this invention provides aneffective bactericidal effect for Salmonella on beef steak.

E. Results with 10 ppm Silver Composition

Results with Silver Composition, in Water.

For Salmonella, for the lower initial bacteria level (10⁴), thefollowing log reductions were recorded: 0.38 at 0 minutes, 0.41 at 20minutes, 0.39 at 60 minutes, and 0.61 at 240 minutes. For the higherinitial bacteria level (10⁴), the following log reductions wererecorded: 0.24 (at 0 minutes, 0.21 at 20 minutes, 0.41 at 60 minutes and0.54 at 240 minutes. The results indicate that the 10 ppm silverembodiment of this invention provides an effective bactericidal effectfor Salmonella on beef steak.

Results with Silver Composition in Water with 10 ppm K₂S₂O₈.

For Salmonella, for the lower initial bacteria level (10⁴), thefollowing log reductions were recorded: 0.26 at 0 minutes, 0.28 at 20minutes, 0.35 at 60 minutes, and 0.58 at 240 minutes. For the higherinitial bacteria level (10⁶), the following log reductions wererecorded: 0.03 at 0 minutes, 0.16 at 20 minutes, 0.21 at 60 minutes and0.36 at 240 minutes. The results indicate that the 10 ppm silver with 10ppm potassium peroxydisulfate (K₂S₂O₈) embodiment of this inventionprovides an effective bactericidal effect for Salmonella on beef steak.

Evidence of Effectiveness of 10 ppm Silver for Treatment of HumanAilments

A. Purpose of Example

The purpose of this example is to demonstrate the utility ofsilver-based composition embodiments of the present invention fortreating a variety of human ailments. The studies in this section wereperformed in Ghana, West Africa, at the Air Force Station Hospital underthe direction of Dr. Kwabiah, at the Korie-Bu Teaching Hospital underthe direction of Sr. Sackey, and at the Justab Clinic/Maternity Hospitalunder the direction of Dr. Abraham. In total, fifty-eight (58) patientswere treated using a composition of the present invention comprising 10ppm silver. The composition was used both internally and externally asan alternative to traditional antibiotics. The ailments treated includedmalaria, upper respiratory tract infections, urinary tract infections,sinusitis, vaginal yeast infections, eye, nose and ear infections, cuts,fungal skin infections, and sexually transmitted diseases, such asgonorrhea.

B. Treatment Methods and Outcomes

Abdominal Pain and Diarrhea.

The method comprises the step of administering approximately 5-25 ml ofsilver composition, one to five times a day orally until there was aresponse. One patient was treated with about 10 ml (about two teaspoons)of a composition of the present invention three times in one day. Thepatient had a full recovery in one day.

Bronchitis.

The method comprises the step of administering ca. 2-25 ml of silvercomposition orally, one to five times a day until there was a response.Two patients were treated with about 5 ml (about one teaspoon) each of acomposition of the present invention for two times a day for three days.The patients had a full recovery in three days.

Vaginal Yeast (Candida).

The method comprises the step of administering ca. 5-25 ml of silvercomposition, one to five times a day as vaginal douches until there wasa response. Five patients were treated with about 10 ml (about twoteaspoons) each of a composition of the present invention for two timesper day. The patients showed a full recovery within six days.

Conjunctivitis.

The method comprises the step of administering ca. several drops of asilver composition, one to five times a day to the infected eye untilthere was a response. Two patients were treated with several drops of acomposition of the present invention in each of the infected eyes fortwo times per day. The patients had a full recovery after one day.

External Cuts and Infection (Including Staphylococcus Skin Infections,Septic Ulcers and Infected Abscesses).

The method comprises the step of administering a silver composition, oneto five times a day to the infected area until there was a response. Sixpatients were treated with about 5 ml (about one teaspoon) each of acomposition of the present invention on the infected areas for two timesper day. The patients showed a full recovery within three days.

External Otitis.

The method comprises the step of administering a silver composition, oneto five times a day to the infected ear until there was a response. Sixpatients were treated with approximately two drops of a composition ofthe present invention into the infected ears for three times per day.The patients showed a full recovery after about four days.

Otitis Media.

The method comprises the step of administering a silver composition, oneto five times a day to the infected ear until there was a response. Onepatient was treated with approximately two drops of a composition of thepresent invention comprising into the infected ear three times per day.The patient showed a full recovery in four days.

Fungal Skin Infection.

The method comprises the step of administering a silver composition, oneto five times a day topically to the infected area until there was aresponse. Two patients were treated with about ten ml (two teaspoons)each of a composition of the present invention three times per day. Thepatients showed a full recovery within eight days.

Gonorrhea.

The method comprises the step of administering a silver composition tothe infected area until there was a response. Two patients were eachtreated with about ten ml (two teaspoons) of a composition of thepresent invention three times per day. The patients showed an absence ofsymptoms within six days.

Malaria.

The method comprises the step of administering a silver composition, oneto five times a day orally to the patient until there was a response.Eleven patients were treated with about ten ml (two teaspoons) each of acomposition of the present invention three times per day. The patientsshowed a resolution of symptoms within five days.

Halitosis and Gingivitis.

The method comprises the step of administering a silver composition, oneto five times a day as a mouthwash until there was a response. Twopatients were each treated with the composition as a mouthwash. Therewas a full resolution of symptoms within three days (gingivitis) andwithin one day (halitosis).

Pelvic Inflammatory Disease.

The method comprises the step of administering about 5-25 ml of silvercomposition, one to five times a day as a vaginal douche until there wasa response. One patient was treated with about 5 ml (approximately oneteaspoon) of a composition of the present invention two times per day.The patient's symptoms resolved within five days.

Pharyngitis.

The method comprises the step of administering a silver composition, oneto five times a day as a gargle until there was a response. Fourpatients were each treated with about ten ml (two teaspoons) of acomposition of the present invention three times per day. The patientsshowed full recovery within six days.

Retrovirus Infection (HIV).

The method comprises the step of administering a silver composition,comprising 5 to 40 ppm silver one to five times a day orally area untilthere was a response. One patient exhibiting HIV (human immunodeficiencyvirus) was treated with about 5 ml (approximately one teaspoon) of acomposition of the present invention two times per day. The patient'ssymptoms resolved within five days.

Sinusitis and Rhinitis.

The method comprises the step of administering a silver composition, oneto five times a day to the nose until there was a response. Six patientswith nasal infections (four with sinusitis and two with rhinitis) wereeach treated with approximately two drops of a composition of thepresent invention comprising in their nasal passages three times perday. The patients showed full recovery within four days.

Tonsillitis.

The method comprises the step of administering a silver composition, oneto five times a day as a gargle until there was a response. One patientwas treated with a composition of the present invention three times perday. The patient showed full recovery within seven days.

Upper Respiratory Tract Infection.

The method comprises the step of administering a silver composition, oneto five times a day orally until there was a response. Two patients wereeach treated with about 5 ml (approximately one teaspoon) of acomposition of the present invention three times per day. The patientsshowed full recovery within six days.

Urinary Tract Infections.

The method comprises the step of administering a silver composition, oneto five times a day orally until there was a response. Three patientswere each treated with about ten ml (two teaspoons) of a composition ofthe present invention two to three times per day. The patients showedfull recovery within six days.

C. Discussion

These results are consistent with the various in vitro tests reportedherein. Essentially, the silver composition is extremely effectiveagainst a large number of microbes in vitro. However, the tests indicatethat this effectiveness remains even in the presence of a large amountof organic material. The silver compositions are widely effective invivo where the organic background is extremely high. Many otherdisinfecting agents are ineffective in the presence of a large amount oforganic material and/or are too caustic or toxic to be used in vivo.

Evidence of Efficacy of 10 ppm Silver Against Tuberculosis Bacteria

A. Purpose

The purpose of this example is to demonstrate the efficacy of a silvercomposition of the present invention against the bacteria that causetuberculosis. This example describes the procedures for evaluation ofthe present invention for tuberculocidal efficacy. The methodology isbased on the Tuberculocidal Activity Test Method as accepted by the EPAon Dec. 11, 1985. (Refer to United States Environmental ProtectionAgency, 1986. Office of Pesticides and Toxic Substances. Data Call-InNotice for Tubercuolocidal Effectiveness Data for All AntimicrobialPesticides with Tuberculocidal Claims. (Received Jun. 13, 1986).

B. Material and Methods

Materials.

The silver composition of the present invention comprised 10 ppm silverin water. The silver composition was evaluated employing a liquid toliquid matrix against Mycobacterium bovis BCG (TMC 1028). This organismcauses tuberculosis in animals and can cause tuberculosis in humans. Itis used as a “stand-in” for M. tuberculosis, the major cause of humantuberculosis, as tests have shown it to have a similar susceptibility toM. tuberculosis. The test organism was exposed to the silver compositionin duplicate at four exposure times and quantified using membranefiltration.

Procedure.

A vial of frozen stock culture was removed from storage and thawed. Anequal volume of buffered gelatin (BUGE) was added to the cell suspensionand homogenized with a Teflon® (brand of polytetrafluoroethylene) tissuegrinder for 1 minute while keeping the culture at 0 to 4° C. in an icebath. The homogenized cell suspension was diluted with saline Tween® 80(brand of polysorbate) solution (ST80) to approximately 10⁷ cfu/ml.

Challenge Titration.

Tenfold serial dilutions of the culture were prepared in dilution blankscontaining 9 ml of neutralizer broth (NEUB) through a 10⁻⁶ dilution.Three 1 ml aliquots of the appropriate dilutions were membrane filteredby first adding 10-20 ml physiological saline solution (PHSS) to thefilter housing and then adding a 1 ml aliquot of the appropriatedilution. The filter was then rinsed with approximately 100 ml of PHSS.The filters were aseptically removed from the filter housing and placedonto 7H11 agar plates. The plates were incubated in a humidified chamberat 37±2° C. for 21 days.

Positive Control.

A tube containing 9 ml of ST80 was prepared and equilibrated to 20±0.5°C. At time 0, 1 ml of test organism culture was added to the tube (1:10dilution). The sample was held for 60 minutes. Tenfold serial dilutionswere prepared in dilution blanks containing 9 ml of NEUB through10⁻⁶-dilution. Three 1 ml aliquots of the appropriate dilutions weremembrane filtered by first adding 10-20 ml PHSS to the filter housingand then adding a 1 ml aliquot of the appropriate dilution. The filterwas rinsed with approximately 100 ml PHSS. The filters were asepticallyremoved from the filter housing and placed onto 7H11 agar plates. Theplates were incubated in a humidified chamber at 37±2° C. for 21 days.

Tests.

Two 25×150 mm tubes containing 9 ml of the test sample were equilibratedto 20±0.5° C. in a water bath. To each tube containing the testdisinfectant (i.e., silver composition), 1 ml of test organism culturewas added. The tube was mixed by swirling and placed back into the waterbath. At 15, 30, 45, and 60 minutes, 1.0 ml aliquots of thedisinfectant-cell suspension were transferred to 9 ml of NEUB and mixedthoroughly. Tenfold serial dilutions were prepared in dilution blankscontaining 9 ml of NEUB through the 10⁻⁶ dilution. Three 1 ml aliquotsof the appropriate dilutions were membrane-filtered by first adding10-20 ml PHSS to the filter housing and then adding a 1 ml aliquot ofthe appropriate dilution. The filter was rinsed with approximately 100ml PHSS. The filters were aseptically removed from the filter housingand placed onto 7HII agar plates. The plates were incubated in ahumidification chamber at 37±2° C. for 21 days.

Phenol Control.

To demonstrate minimum culture viability and resistance, the culture wastested against a 0.8% phenol solution. A 1 ml aliquot of test organismculture was placed into 9 ml of the phenol solution equilibrated to25±0.5° C. and incubated for 20 minutes. After the exposure period, 1 mlfrom the phenol/organism solution was removed and added to 9 ml of NEUB.Tenfold serial dilutions were prepared in dilution blanks containing 9ml of NEUB through 10⁻⁶ dilution. Three 1 ml aliquots of the appropriatedilutions were membrane filtered by first adding 10-20 ml PHSS to thefilter housing and then adding a 1 ml aliquot of the appropriatedilution. The filter was rinsed with approximately 100 ml PHSS. Thefilters were aseptically removed from the filter housing and placed onto7H11 agar plates. The plates were incubated in a humidified chamber at37±2° C. for 21 days.

Neutralization Verification.

A 1 ml aliquot of the disinfectant was added to 8 ml of NEUB. Thedisinfectant/neutralizer broth was allowed to equilibrate to the sametemperature as the test samples. One ml of test organism culture wasadded to the mixture and mixed thoroughly. Incubation was continued forthe approximate time it would take to filter a sample. Additionally, a 1ml aliquot of test organism was added to 9 ml of NEUB and mixedthoroughly (1:10 dilution). Tenfold serial dilutions of both tubes wereprepared in dilution blanks containing 9 ml of NEUB thought 10⁻⁶dilution. Three 1 ml aliquots of the appropriate dilutions were membranefiltered by first adding 10-20 ml PHSS to the filter housing and thenadding a 1 ml aliquot of the appropriate dilution. The filter was rinsedwith approximately 100 ml PHSS. The filters were aseptically removedfrom the filter housing and placed on 7H11 agar plates. The plates wereincubated in a humidified chamber at 37±2° C. for 21 days.

C. Results

The starting titer for the challenge culture was 4.7×10⁷ cfu/ml. Thepositive control titer was 6.5×10⁶ cfu/ml. The media used in this studyeffectively demonstrated neutralization with a 95.2% recovery in adisinfectant/neutralizer solution when compared to a media blank.

For the test plates, expected counts were underestimated and thereforethe reported counts exhibit “>” to mark that the count is an estimationand that accurate counts are beyond the limit of detection for thedilutions plated.

In calculating the log and percent reductions of the disinfectantagainst M. bovis, the estimated counts which have “greater than” countsresulted in “less than” log and percent reductions (“<”). The purpose ofthis is to demonstrate that the results are an estimation and beyond theaccurate limit of detection for the dilutions plated. All reductionswere calculated using the positive control as the initial starting titerof the organism. The results for log and percent reductions aresummarized below. As a measure of the resistance of the challengeculture, the phenol resistance of the M. bovis showed a ˜1.81 logreduction with 20 minutes of exposure to 0.8% phenol.

Replicate One:

Exposure time Log reduction Percent reduction 15 minutes <0.12 <12.3% 30minutes <0.22 <40.0% 45 minutes <1.57 <97.2% 60 minutes <1.56 <97.2%

Replicate Two:

Exposure time Log reduction Percent reduction 15 minutes <0.26 <44.8% 30minutes <0.20 <36.9% 45 minutes <1.58 <97.3% 60 minutes <1.53 <97.1%

D. Conclusions

The use of silver compositions of the present invention is effectiveagainst tuberculosis bacteria. A method comprising the step ofadministering silver compositions of the present invention is effectiveagainst tuberculosis organisms.

Evidence of Efficacy of 10 Ppm Silver Against Candida Albicans ATCC#10231, Trichomonas Vaginalis ATCC #20235, and MRSA StaphyloccocusAureus ATCC #BAA-44

A. Purpose of Example

The purpose of this example is to illustrate the efficacy of silvercompositions of the present invention against Candida albicansATCC10231, Trichomonas vaginalis ATCC 20235, and drug resistantStaphylococcus aureus ATCC BAA-44.

Candida albicans, a yeast, and Trichomonas vaginalisis, a protozoa, cancause numerous health problems including vaginal infections, diaperrash, and thrush. The results below show that silver compositions of thepresent invention produced nearly a 100% kill of both organisms. Theresults show the utility of silver compositions of the present inventionin a feminine hygiene product and in a diaper rash product.

Staphylococcus aureus can cause serious blood poisoning when it enters awound. It once was easily treated with penicillin, but the organism hasnow mutated to the point where it is totally resistant to penicillin.The next defense on the antibiotic ladder has been methicillin, butmethicillin-resistant strains have become increasingly common,especially in hospitals. These strains are known as MRSA(methicillin-resistant Staphylococcus aureus) and have been dubbed the“superbug.” People who contract MRSA can die in a matter of days. In theresults reported in this example, a silver composition of the presentinvention was found to kill 91.6% of the MRSA in just 10 minutes, and99.5% in an hour. The results show the utility of silver compositions ofthe present invention in killing MRSA, a known infectious threat.

B. Methods and Results

Employing the USP Preservative Rapid Challenge Test with a compositionof the present invention comprising 10 ppm silver in water, thefollowing results were obtained. These results show that silvercompositions of the present invention can be effective against yeastinfections, protozoa infections, and drug resistant bacteria infections.

Candida albicans ATCC #10231.

The initial concentration of Candida albicans yeast was 6.8×10⁵ cfu/ml.After contact for either 10 minutes, 30 minutes, 1 hour, or one day withthe silver composition, there were no colonies detected.

Trichomonas vaginalis ATCC #30235.

The initial concentration of Trichomonas vaginalis protozoa was 6.0×10⁴cfu/ml. After contact with the silver composition for either 10 minutes,30 minutes, 1 hour, or one day, there was 0% motility of 100 Organisms.That is, one hundred (100) Trichomonas vaginalis parasites were analyzedvia microscopy for motility of flagella. None of the one-hundred (100)parasites demonstrated motility after only ten (10) minutes of contactwith the silver composition indicating inhibitory or lethal propertiesof the silver composition on the parasites. The outer membranes oftwenty-five (25) percent of the parasites had ruptured after contact ofone (1) day.

Staphylococcus aureus MRSA ATCC #BAA-44.

The initial concentration of methicillin-resistant Staphylococcus aureus(MRSA) was 6.0×10⁶ cfu/ml. After contact with the silver composition,there were 500,000 cfu/ml detected after 10 minutes contact (91.6%killed), 70,000 cfu/ml after 30 minutes contact (98.8% killed), 30,000cfu/ml after 1 hour contact (99.5% killed), and fewer than 10 cfu/mlafter one day contact (virtually total kill).

Evidence of the Efficacy and Lack of Cytotoxicity of 10 ppm Silver, 14ppm Silver+1.5% H₂O₂, and 22 ppm Silver in Inhibiting DNA Polymerase andReverse Transcriptase in the Context of Hepatitis B

A. Purpose of Example

The purpose of the example is to illustrate the efficacy of silvercompositions of the present invention against hepatitis B. This exampleshows that silver compositions of the present invention have antiviralproperties. Any agent used in antiviral therapy should exhibit little orno cytotoxicity so cytotoxicity of the silver compositions was analyzed.

Hepatitis B is caused by a DNA virus of the hepadnaviridae family ofviruses. The Hepatitis B Virus (HBV) is a 3.2 kb DNA virus, replicatingalmost exclusively in the liver cells (hepatocytes). Replicationinvolves two main enzymes: DNA polymerase and reverse transcriptase. Theresults of this example show that silver compositions of the presentinvention interfere with replication involving either DNA polymerase orreverse transcriptase. The results of this example show that silvercompositions of the present invention have antiviral properties. Theresults of this example show that silver compositions of the presentinvention can be effective against hepatitis B.

As further detail, when hepatitis B enters the body of a new host, itinfects the liver if it gets past the host's immune system. In theinfection, the virus attaches to the membrane of a liver cell, and thecore particle of the virus enters the liver cell. The core particle thenreleases its contents of DNA and DNA polymerase into the liver cellnucleus. Within the liver cell, the virus replicates via reversetranscription and translation processes, which involve reversetranscriptase and DNA polymerase enzymes. The DNA polymerase causes theliver cell to make copies of hepatitis B DNA. These copies of the virusare released from the liver cell membrane into the blood stream. Fromthere, they can infect other liver cells and thus replicate effectively.The incubation period of the hepatitis B virus is about 6 to 25 weeks(i.e., time before physical and generally detectable histological orphysical symptoms occur). However, there are several biochemical andhistological changes that occur in the early stages following infectionwith the hepatitis B virus.

B. Materials

Solutions comprising 10 ppm, 14 ppm, 22 ppm, and 32 ppm silvercompositions according to the present disclosure were used. Thenucleotides dATP, dGTP, dCTP, and [³H]-dTTP were obtained from standardcommercial sources, as were the compounds lamivudine (a syntheticantiretroviral agent) and zidovudine (AZT). Isolated Hepatitis B viruswas freshly obtained from a person suffering from Hepatitis B infectionand was taken up by Haffine Institute, Mumbai INDIA (a WHO certifiedtesting laboratory). Test cell cultures (Vero and Hep2) were grown asconfluent monolayers by typical cell culture methods.

C. Methods

1) Procedure for Test of DNA Polymerase Inhibition.

Overall Approach.

Hepatitis B viral extracts from human subjects are incubated withradiolabelled nucleotides and an active inhibitor. Percent inhibition iscalculated based on the amount of de novo viral nucleic acid synthesizedwith respect to lamivudine as a positive control and phosphate buffersaline (PBS) as a negative control.

Specific Procedure.

Isolated Hepatitis B virus was lysed to extract free polymerase enzyme,which is free from contaminating enzymes. A virus extract (25 μl) wasadded to a reaction mixture comprising dATP, dGTP, dCTP and [³H]dTTPnucleotides (25 μl). Active inhibitor (3 μl) was added to the mixturecomprising virus extract and nucleotides. The resultant mixture wasincubated at 37° C. for 2 hours.

A separate negative control experiment was performed in which phosphatebuffer saline (PBS, 3 μl) was used instead of the inhibitor (3 μl).

A separate positive control experiment was performed in which a knownDNA polymerase inhibitor (3 μl of lamivudine at a concentration 3 mg/ml)was used instead of the tested inhibitor (3 μl).

The reaction was stopped by adding 25 μl EDTA and 25 μl TCA(trichloroacetic acid). The reaction mixture was then spotted on ionicpaper (DEAE paper). The paper was washed three times with TCA and thenwith ethyl alcohol. The filter paper was air dried and put into ascintillation vial with a scintillation cocktail. Radioactivity wasmeasured by a liquid scintillation counter (Blue Star). As a countingcontrol, a blank silver composition was run through the completeprocedure without viral load, to check any potential interference in thescintillation counter method.

A reference for this method is P. S. Venkateswaran, I. Millman, and B.S. Blumberg, “Effect of an extract from Phyllanthus niruri on hepatitisB and woodchuck hepatitis viruses: in vitro and in vivo studies,” Proc.Natl. Acad. Sci., USA, 1987, 84, 274-278, which is incorporated hereinby reference.

2) Procedure for Test of Reverse Transcriptase Inhibition.

A commercial viral enzyme preparation of Moloney murine leukemia virusreverse transcriptase (MoMuLV) having Poly(A)dT (primer for RT) wasused. 50 μl of the MoMuLV preparation was combined with a mixture ofdATP, dGTP, dCTP and [³H]dTTP nucleotides.

This mixture was combined with 3 μl of the inhibitor to be tested, andthe resultant mixture was incubated at 37° C. for 2 hours.

A negative control experiment was performed in which phosphate buffersaline (PBS, 3 μl) was used instead of the inhibitor.

A positive control experiment was performed in which a known reversetranscriptase inhibitor (3 μl of AZT at a concentration 0.625microgram/ml) was used instead of the tested inhibitor.

The reaction was stopped by adding 25 μl EDTA and 25 μl TCA. Thereaction mixture was then spotted on ionic paper (DEAE paper). The paperwas washed three times with TCA and then with ethyl alcohol. The filterpaper was air dried and put in a scintillation vial with a scintillationcocktail. Radioactivity was measured by a liquid scintillation counter(Blue Star).

3) Procedure for Testing Cytotoxicity.

Cells were prepared from healthy, confluent Vero and Hep2 cell culturesthat were maintained by passage every 3-4 days. One day prior to thetest cells were released from the cultures using standard techniques andsuspended in a growth medium and dispensed into wells of a microtiterplate and placed in a 5% CO₂ incubator at 37±2° C. An aliquot (100 μl)of each test substance was introduced into a well (in triplicate) with100 μl of PBS as a control. Every 24 hrs the wells were examined underhigh power of an inverted microscope to check for any cytopathic effect(CPE).

D. Results

Results for Test of Reverse Transcriptase Inhibition:

Sample % Inhibition negative control (PBS) 0 positive control (AZT)31.33 Silver, 10 ppm 89.52 Silver, 14 ppm and 1.5% H2O2 86.93 Silver, 22ppm 84.46

Results for Test of DNA Polymerase Inhibition:

Sample % Inhibition negative control (PBS) 0 positive control(lamivudine) 31.33 Silver, 10 ppm 77.73 Silver, 14 ppm with 1.5% H2O265.6 Silver, 22 ppm 60.89

Silver Compositions of the Present Invention are Highly Effective atInhibiting DNA Polymerase

Results for Test of Reverse Transcriptase Inhibition:

Sample % Inhibition negative control (PBS) 0 positive control (AZT)18.06 Silver, 10 ppm 89.52 Silver, 14 ppm with 1.5% H2O2 86.93 Silver,22 ppm 84.46

Thus, silver compositions of the present invention inhibit reversetranscriptase. Silver compositions of the present invention would beexpected to be effective against human ailments propagated by viruses,such as hepatitis B.

Results for Test of Cytotoxicity:

Sample Vero Hep2 control (PBS) No CPE No CPE Silver, 10 ppm No CPE NoCPE Silver, 14 ppm with 1.5% H2O2 CPE positive CPE positive Silver, 22ppm No CPE No CPE

These results indicate that the silver composition is essentiallynon-cytotoxic. As expected, hydrogen peroxide, which is known to becytotoxic, shows a cytotoxic effect. Thus, the silver should be harmlessto cells when used in vivo.

12. Evidence of Efficacy of Silver Composition as Water Disinfectant

A. Purpose

Tests were carried out to demonstrate the efficacy of the inventivecomposition in disinfecting drinking water.

B. Methods

A sample of raw river water was spiked with two loopfuls of Klebsiellaoxtyoca. 100 ml aliquots of this of this spiked water solution werebrought to 0.05 ppm, 0.1 ppm, 0.2 ppm, 0.5 ppm, or 1.0 ppm of inventivesilver composition. After an incubation of 5-60 minutes, the sampleswere membrane filtered. The filter was rinsed with approximately 100 mlsterile water. The filters were aseptically removed from the filterhousing and placed on coliform nutrient agar plates. The plates wereincubated under growth conditions for 24 hours and counted.

Total Coliform Sample Silver (ppm) Contact (min) (per ml) Cfu/100 ml rawwater — — 36 TNTC 1 1.00 5.0 0 0 2 1.00 10.0 0 0 3 1.00 15.0 0 0 4 1.0030.0 0 0 5 0.50 10.0 0 0 6 0.50 30.0 0 0 7 0.50 60.0 0 0 8 0.20 5.00 0 09 0.20 10.0 0 0 10 0.20 30.0 0 0 11 0.20 60.0 0 0 12 0.10 10.0 0 0 130.05 20.0 0 0 TNTC = too numerous to count.

The silver composition proved to be surprisingly effective. Even at theshortest time (20 min) allowed for incubation of the lowestconcentration tested (0.05 ppm) there was a complete kill of thebacteria. At 0.20 ppm and higher there was a complete kill at 5 minutes.It seems clear that a complete kill takes less than 5 minutes.

Evidence of Efficacy of 32 ppm Silver as Surface Disinfectant

The Environmental Protection Agency (EPA) has approved a 32 ppm silvercomposition of the present invention as a broad spectrum surfacedisinfectant for use in hospitals, medical environments, residentialhomes, commercial buildings, and businesses. It has been approved foruse against some of the most deadly pathogens including: Gram-positivebacteria, such as Staphylococcus aureus (presently considered to be themost deadly bacteria in U.S. hospitals), Gram-negative bacteria, such asSalmonella choleraesuis (responsible for food poisoning), and nosocomialor hospital-acquired pathogens, such as Pseudomonas aeruginosa (oftenfound in burns and cuts).

Silver compositions of the present invention can be sprayed in andaround occupied areas without endangering the lives of people. One candisinfect surfaces selected from the group consisting of walls, tables,chairs, light fixtures, bathrooms, glass, porcelain, metal, glazedceramic, enameled and painted by means of spraying or by means of wipingwith a silver composition of the present invention. A preferred methodof disinfecting comprises one or more of the steps of cleaning thesurface to be disinfected, applying, by means of a spray, mop, sponge,or cloth, a composition of the present invention, thoroughly wetting thearea to be disinfected, allowing the surface to remain wet for at least10 minutes at a temperature of at least 20° C. (time/temperatureinterrelation can be adjusted via the Arrhenius equation or other meansknown to one of ordinary skill), and wiping the surface with a cleanpaper or cloth towel. Compositions for disinfecting surfaces comprisethose comprising 5 to 40 ppm silver. A preferred composition of thepresent invention for disinfecting surfaces comprises (32±3) ppm silver.Another preferred composition of the present invention for disinfectingsurfaces comprises (10±2) ppm silver. Another preferred composition ofthe present invention for disinfecting surfaces comprises (22±2) ppmsilver.

Evidence of Efficacy of Silver Composition as Super Disinfectant

A. Purpose of Example

The purpose of this example is to show the antimicrobial activity of asilver composition of the present invention (here 10 ppm silver, 14 ppmsilver with 1.5 wght % hydrogen peroxide, and 32 ppm silver) against thetest organism Yersinia pestis, the etiologic agent of bubonic plague. Byperforming a standard kill-time assay using a Y. pestis suspension, itis demonstrated that silver compositions of the present invention areeffective even against the bubonic plague bacteria.

B. Material and Methods

Y. Pestis, strain D27, was grown on a Columbia Agar plate for about 24hours at 30° C. in a 5% CO₂ incubator. Growth from the plate was scrapedinto suspension, using 3 ml of sterile HPLC water. The suspension wastransferred to a 50 ml conical centrifuge tube. The plate was thenrinsed using an additional 2 ml of HPLC water. This rinse was added tothe centrifuge tube. The tube was centrifuged at 3,500×g for 5 minutes.The supernatant was discarded and the pellet was resuspended in 1 ml ofHPLC water, to give a final concentration of approximately 10¹⁰ cellsper ml.

The method involved the following steps:

1 A 9.9 ml aliquot of the silver composition to be tested was placed ina sterile 20 mm×150 mm tube. The tube was equilibrated in a 20° C. waterbath.

2 The tube of silver composition was inoculated with 100 μl of the testorganism suspension at time zero to form a mixture. The tube wasimmediately vortexed and returned to the water bath.

3 At 2 min, 3 min, 4 min, and 5 min for 10 ppm or 32 ppm silver or 2min, 4 min, 6 min and 8 min for 14 ppm silver with 1.5% v/v H₂O₂, 1 mlof organism/silver mixture was removed to 99 ml of neutralizer in a 250ml Erlenmeyer flask. The flask was mixed thoroughly.

4 The neutralized suspension was immediately serially diluted 1:10 inphysiological saline solution (PSS).

5 The number of viable organisms in selected dilution tubes and flaskswas assayed by membrane filtration. One ml aliquots were plated induplicate. The membranes were washed with about 150 ml (or 250 ml if thesample was taken from the neutralizer flask) of sterile phosphatebuffered saline and removed to Columbia Agar plates. The entireremaining contents (98 ml) of the 4 & 5 min neutralizer flasks were alsoplated. The plates were incubated at 30° C. in a 5% CO₂ incubator for 72hours.

6 The number of colonies on each filter was counted and log reductionswere computed.

C. Results

The results for 10 ppm silver are as follows:

Time Log Reduction Percent Kill 2 min 2.63 99.77 4 min 3.20 99.94 6 min3.46 99.97 8 min 3.68 99.98

The calculated regression equation for these data is Y=2.3965+0.1696x.This indicates that the time for a 6-log reduction is 21.2 minute.

The results for 32 ppm silver are as follows:

Time Log Reduction Percent Kill 2 min >7.61 99.999998 4 min >7.6199.999998 6 min >7.61 99.999998 8 min >7.61 99.999998

The results for 14 ppm silver with 1.5% v/v H₂O₂ are as follows:

Time Log Reduction Percent Kill 2 min 3.27 99.95 3 min 4.72 99.998 4 min5.36 99.9996 5 min 6.47 99.99997

The calculated regression equation for these data is Y=1.371+1.024x.This indicates that the time for a 6-log reduction is 4.52 minute.

The silver composition of the present invention exhibited significantbactericidal activity against Y. pestis, the etiologic agent of bubonicplague. The 32 ppm composition gave more than a 7 log reduction(essentially total kill) in less than 2 min. The data show that the 10ppm silver takes some 20 min to achieve a 6 log kill. The silver andhydrogen peroxide show significant synergism with a calculated 6 logkill of under 5 min. This is much better than 10 ppm silver alone. Thelevel of 14 ppm silver was chosen because the data of other experimentssuggested that this level of silver combined with hydrogen peroxidewould achieve results approaching those of the 32 ppm silver product.

Data Summary

The following table contains a summary of the above results in terms ofthe effects of the inventive silver composition on a wide variety ofmicrobes and human diseases. In some cases, the data presented in thetable is not repeated above. However, the results were obtained usingthe procedures explained above so that one of ordinary skill in the artcan readily replicated the results.

Human Diseases Cured by and Pathogens Killed by the Inventive SilverComposition:

Effective Disease Pathogen Concentration Boils Staphylococcus aureusKilled @ 5 ppm Osteomyelitis Staphylococcus aureus Killed @ 5 ppmBacillary Shigella boydii Killed @ 2.5 ppm Dysentery Burn InfectionsPseudomonas aeruginosa Killed @ 5 ppm Dental Plaque Streptococcus mutansKilled @ 5 ppm Diarrhea (Bloody) Shigella boydii Killed @ 2.5 ppmDiarrhea Escherichia coli Killed @ 2.5 ppm Ear Infection Haemophilusinfluenzae Killed @ 1.25 ppm Ear Infection Streptococcus pneumonieKilled @ 2.5 ppm Enteric Fever Salmonella tyhimurium Killed @ 2.5 ppmEpiglottitis (In Haemophilus influenzae Killed @ 1.25 ppm children) EyeInfections Staphylococcus aureus Killed @ 5 ppm Corneal Ulcers-Pseudomonas aeruginosa Killed @ 5 ppm Keratitis Food PoisoningSalmonella arizona Killed @ 5 ppm Food Poisoning Salmonella tyhimuriumKilled @ 2.5 ppm Food Poisoning Escherichia coli Killed @ 2.5 ppmEndocarditis Streptococcus faecalis Killed @ 2.5 ppm EndocarditisStreptococcus gordonii Killed @ 5 ppm Meningitis Haemophilus influenzaeKilled @ 1.25 ppm Meningitis Enterobacter aerogenes Killed @ 2.5 ppmMeningitis Pseudomonas aeruginosa Killed @ 5 ppm MeningitisStreptococcus pneumonie Killed @ 2.5 ppm Nosocomial Klebsiellapneumoniae Killed @ 2.5 ppm Infections Nosocomial Pseudomonas aeruginosaKilled @ 5 ppm Infections Nosocomial Streptococcus pyogenes Killed @1.25 ppm Infections (From hospitals) Pneumonia Staphylococcus aureusKilled @ 5 ppm Pneumonia Haemophilus influenzae Killed @ 1.25 ppmPneumonia Pseudomonas aeruginosa Killed @ 5 ppm Pneumonia Streptococcuspneumonie Killed @ 2.5 ppm Respiratory Tract Streptococcus pyogenesKilled @ 1.25 ppm Infections Respiratory Tract E. coli Killed @ 2.5ppm,, Infections

Effective Disease Pathogen Concentration Respiratory Tract Klebsiellapneumoniae Killed @ 2.5 ppm Infections Scarlet Fever Streptococcuspyogenes Killed @ 1.25 ppm Septicemia Enterobacter aerpyogenes Killed @2.5 ppm Sinus Infections Haemophilus influenzae Killed @ 1.25 ppmSinusitis Streptococcus pneumonie Killed @ 2.5 ppm ImpetigoStaphylococcus aureus Killed @ 1.25 ppm Skin Infections Staphylococcusaureus Killed @ 5 ppm Skin Infections Streptococcus pyogenes Killed @1.25 ppm Strep Throat Streptococcus pyogenes Killed @ 1.25 ppmSuppurative Haemophilus influenzae Killed @ 1.25 ppm Arthritis ThroatInfections Haemophilus influenzae Killed @ 1.25 ppm Tooth DecayStreptococcus mutans Killed @ 5 ppm Urethritis (Men) Trichomonasvaginalis Killed @ 10 ppm Urinary Tract E. coli Killed @ 2.5 ppmInfections Urinary Tract Klebsiella pneumonias Killed @ 2.5 ppmInfections Urinary Tract Pseudomonas aeruginosa Killed @ 5 ppmInfections Urinary Tract Streptococcus faecalis Killed @ 2.5 ppmInfections Urinary Tract Enterobacter aerpyogenes Killed @ 2.5 ppmInfections Vaginitis (Women) Trichomonas vaginalis Killed @ 10 ppm WoundInfections Escherichia coli Killed @ 2.5 ppm Wound InfectionsEnterobacter aerpyogenes Killed @ 2.5 ppm Wound Infections Klebsiellapneumoniae Killed @ 2.5 ppm Wound Infections Pseudomonas aeruginosaKilled @ 5 ppm Wound Infections Streptococcus faecalis Killed @ 2.5 ppmYeast Infections Candida albicans Killed @ 10 ppm

Efficacey of Silver Colloid Formulated as a Hydrogel

Modern wound care has come to recognize the fact that for optimalhealing a wound should be kept sterile and protected from desiccationand environmental contaminants. Traditional bandages are effective asproviding protection from environmental contaminants but are largelyineffective at preventing desiccation. Bandages may be renderedantimicrobial through the addition of a variety of disinfectantsubstances, but these substances are often harsh and kill cells or thebody as well as microbes. In recent times wound care has beenrevolutionized by hydrogel materials which are available as either asemisolid (amorphous material) or as a soft sheet-like material. Thehydrogel is hydrophilic and hence prevents desiccation of the wound. Thesheet-like material is effective at excluding environmental contaminantsand because of its hydrophilic character, the hydrogel can actuallyabsorb excess fluid exuded by the wound.

Hydrogels are formed by combining a hydrophilic polymer with otheringredients in an aqueous solution. The polymer forms a gel following achange in pH, temperature or other triggering event. In a gel a finemolecular network of the polymer surrounds regions of the aqueoussolution. Although the composition may be an amorphous semi-solid or afirmer sheet-like material, the vast majority of the volume tends to beoccupied by the aqueous solution as opposed to the hydrophilic polymer.Hydrophilic polymers that are appropriate for the production ofhydrogels include gelatin, carboxy-methyl cellulose (and other cellulosederivatives), other carbohydrate polymers of plant or algal origin suchas alginate, carrageenan, xanthan gum, locust bean gum, gum traganth,guar gum, gum arabic and other plant gums, acrylic acid copolymers (suchas Carbopol), and combinations of these and similar hydrophilicpolymers.

The aqueous component preferably contains various additive substancesthat enhance the physical characteristics of the hydrogel and/or enhancewound healing. These include various vitamins, amino acids and growthfactors added to enhance healing or reduce scar formation to diminishscarring. Common anesthetics such as novocaine, lidocaine andderivatives thereof can also be incorporated as additives to enhancecomfort. Since keeping the wound sterile is a major goal of thedressing, various antimicrobial or disinfectant agents areadvantageously included. These include organic acids such as citricacid, dilute acetic acid, benzoic acid, proprionic acid and lactic acid.Alcohols such as isopropanol or ethanol are useful as are organicdisinfectants including chlorinated phenolics such as “TCP” (2,4,6trichlorophenol), biguanides, chlorhexidine (when mixed with cetrimide),chlorhexidine gluconate, and chlorhexidine acetate. Disinfectantsurfactants including amphotheric surfactants and aldehydes such asformaldehyde and glutaraldehyde can be included. Halogen disinfectantsincluding iodine, iodophores, and polyvidone-iodine are effective as areperoxides and other oxygenators such as hydrogen peroxide. Otherbeneficial ingredients include aluminum-zinc astringent agents, furanderivatives and quinoline derivatives such as clioquinol. As beneficialas all these antimicrobial agents may be, they all tend to suffer fromthe defect that they can be damaging to tissue and/or microbes canreadily develop resistance to them.

As amply demonstrated above, the inventive silver colloid is highlyeffective antimicrobially, is very gentle to human tissue and iseffective against resistant microbes. Both amorphous gel and hydrogelsheet are both amenable to delivering effective levels of colloidalsilver in moist healing environment. On one hand the amorphous hydrogelslowly releases colloidal silver as it slowly softens in tissue exudateand gradually begins to dissolve. On the other hand amorphous hydrogeldonates moisture to the tissue and simultaneously makes colloidal silveravailable at site. In addition, a small amount of colloidal silverpresent in the dressing has the advantage of being molecular silver,whose gradual reduction over an extended period of time will releasesilver ions which have excellent oligodynamic activity.

After initial experiments Carbopol was selected as an effective hydrogelforming agent for use with the inventive colloidal silver. A basicformulation was developed which generally included the followingingredients:

Ingredient Function Supplier Colloidal Silver Solution Active,Anti-microbial & American (22 ppm or 32 ppm) Diluent Biotech LabsCarbopol ETD2020 Rheology Modifier Noveon Triethanolamine Neutralizer,Penetrating E. Merck agent Propylene Glycol Humectant E. Merck

All raw materials were first analyzed for

1. Anti-bacterial Activity

2. Physical & Chemical Properties:

-   -   1. Appearance    -   2. Odor    -   3. pH    -   4. Feel    -   5. Density    -   6. Foaming Property    -   7. Flow-ability.

Colloidal Silver Solution (22 ppm or 32 ppm):

In this formulation Silver Solution is used as an active component(anti-microbial agent). It is also the only diluent in this specificformulation.

A. Anti-Bacterial Activity:

Diameter of zone of inhibition Culture 22 ppm 32 ppm MRSA 17 mm 18 mm E.coil 14 mm NA Ps. aeruginosa 21 mm 22 mm

B. Physical & Chemical Properties:

1. Appearance Colorless clear liquid 2. Odor Odorless 3. pH 5.0 4. FeelNot Applicable 5. Density 1.00 6. Foaming Property Not Applicable 7.Flow-ability Not Applicable

Carbopol

Carbopol is chemically known as carboxypolymethylene or carboxyvinylpolymer. It is a copolymer of acrylic acid and is highly ionic (i.e.,hydrophylic) and slightly acidic compound. Carbopol polymers must beneutralized in order to achieve maximum viscosity. It is used inpharmaceuticals, cosmetic & textile printing fields as a thickening,suspending, dispersing and emulsifying agent. In this formulationCarbopol is used as a gelling or thickening agent.

A. Anti-Bacterial Activity Not Applicable

B. Physical & Chemical Properties:

1. Appearance Dry, white powder 2. Odor Odorless 3. pH Not Applicable 4.Feel Not Applicable 5. Density Not Applicable 6. Foaming Property NotApplicable 7. Flow-ability Not Applicable

Triethanolamine

(TEA) C₆H₁₅NO₃ ₍Mol. Wt.: 149.19)

In this formulation Triethanolamine, an alkalizing agent neutralizesCarbopol to raise the viscosity. It also increases the penetrating powerof the active agent.

A. Anti-Bacterial Activity (Not Applicable)

B. Physical & Chemical Properties

1. Appearance Colorless viscous liquid 2. Odor Slight ammoniacal 3. pHNot Applicable 4. Feel Not Applicable 5. Density 1.1242 g/cc 6. FoamingProperty Not Applicable 7. Flow-ability Not Applicable

Propylene Glycol

C₃H₈O₂ Mol. Wt.: 76.09

Propylene Glycol is chemically known as 1:2 propanediol. It is used as ahumectant and feel modifier in this formulation.

A. Anti-Bacterial Activity Not Applicable

B. Physical & Chemical Properties:

1. Appearance Colorless viscous liquid 2. Odor Odorless 3. pH NotApplicable 4. Feel Not Applicable 5. Density 1.036 gm/cc 6. FoamingProperty Not Applicable 7. Flow-ability Not Applicable

Once the standard formula was developed a number of batches weremanufactured to explore the possible range of formulations. From the 19experiments carried out the following observation were reached.

1. Increase in pH increases viscosity of gel.

2. Increase in Carbopol quantity increases viscosity of gel.

3. Higher the Carbopol percentage the higher the tackiness.

From the above experiments it can be concluded that a trade off has tobe reached between amount of Carbopol & TEA used and the final pHobtained which should not be more than 8.5. Hence formulation No. 18 waskept as standard and batch scaled up to 10 Kg.

Product Development Studies Introduction:

Carbopol based gel formulations have to be standardized with respect topH, feel, tackiness and consistency. With this in mind various labbatches were taken using water as the aqueous phase to obtain a productof suitable quality and feel before taking the main batch.

Batch No. SG/001 Formulation: Part A: Distilled Water 83.50 g  Carbopol00.62 g  NaOH 18% 00.60 g  Part B: Distilled Water 1.00 g Propyleneglycol 5.00 g NaOH 18% 1.50 g

Procedure: Weigh the given amount of Distilled Water from Part A andkeep in water bath at 70° C. Add Carbopol to Distilled Water withconstant stirring to avoid lumps. Add NaOH 18% to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results: 1. pH 10.8 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min.3. Tackiness Very Tacky Batch No. SG/002 Formulation: Part A: Distilledwater 83.50 g Carbopol 00.62 g TEA 01.20 g Part B: Distilled water  1.0g Propylene glycol  5.0 g TEA  1.5 gm

Procedure: Weigh the given amount of Distilled water from part A andkeep in water bath at 70° C. Add Carbopol to Distilled water withconstant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from part B and keep in water bath at70° C. for 15-20 min. Add part B to part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 7.9 (SOP-08) 2. Flow-ability 90° C. => >5 min 45° C. => >5min 3. Tackiness Very Tacky Batch No. SG/003 Formulation: Part A:Distilled water 86.00 g Carbopol 00.62 g TEA 01.20 g Part B: Distilledwater  2.00 g Propylene glycol  5.00 g TEA  1.50 g

Procedure: Weigh the given amount of Distilled water from part A andkeep in water bath at 70° C. Add Carbopol to Distilled water withconstant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from part B and keep in water bath at70° C. for 15-20 min. Add part B to part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 8.62 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min 3.Tackiness Very Tacky. Batch No. SG/004 Formulation: Part A: Distilledwater 86.00 g Carbopol 00.62 g TEA 01.00 g Part B: Distilled water  2.00g Propylene glycol  5.00 g TEA  1.50 g

Procedure: Weigh the given amount of Distilled water from Part A andkeep in water bath at 70° C. Add Carbopol to Distilled Water withconstant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results: 1. pH 8.5 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min.3. Tackiness Very Tacky Batch No. SG/005 Formulation: Part A: Distilledwater 86.0 g Carbopol 0.62 TEA 1.20 g Part B: Distilled water 2.00 gPropylene glycol 7.00 g TEA 1.50 g

Procedure: Weigh the given amount of Distilled Water from Part A andkeep in water bath at 70° C. Add Carbopol to Distilled Water withconstant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 8.7 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min. 3.Tackiness Very Tacky Batch No. SG/006 Formulation: Part A: Distilledwater 85.00 g Carbopol 00.62 g TEA 01.00 g Part B: Distilled water  1.00g Propylene glycol  5.00 g TEA  1.40 g

Procedure: Weigh the given amount of Distilled Water from Part A andkeep in water bath at 70° C. Add Carbopol to Distilled Water withconstant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 8.4 2. Flow-ability 90° C. => >5 min 45° C. => >5 min 3.Tackiness Very Tacky Batch No. SG/007 Formulation: Part A: DistilledWater  172 g Carbopol 1.24 g TEA 2.40 g Part B: Distilled Water  6.0 gPropylene glycol   10 g TEA 2.80 g

Procedure: Weigh the given amount of Distilled Water from Part A andkeep in water bath at 70° C. Add Carbopol to Distilled Water withconstant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 8.28 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min.3. Tackiness Very Tacky Batch No. SG/008 Formulation: Part A: SilverSolution (32 ppm)   86 g Carbopol 0.62 g TEA 1.20 g Part B SilverSolution (32 ppm)  2.0 g Propylene glycol  5.0 TEA  1.5 g

Procedure: Weigh the given amount of Silver Solution from part A andkeep in water bath at 70° C. Add Carbopol to Silver Solution withconstant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 8.65 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min.3. Tackiness Very Tacky Batch No. SG/009 Formulation: Part A: SilverSolution (32 ppm)  172 g Distilled Water   12 g Carbopol 1.24 g TEA 2.40g Part B: Silver Solution (32 ppm) 6.00 g Propylene glycol 10.0 g TEA2.80 g

Procedure: Weigh the given amount of Silver Solution from part A andkeep in water bath at 70° C. Add Carbopol to Silver Solution withconstant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results: 1. pH 8.54 2. Flow-ability: 90° C. => >5 min. 45° C. => >5 min.3. Tackiness Very Tacky Batch No. SGI010 Formulation: Part A: SilverSolution (32 ppm)  172 g Distilled Water   24 g Carbopol 1.39 g TEA 2.40g Part B: Silver Solution (32 ppm) 6.00 g Propylene glycol 5.00 g TEA2.80 g

Procedure: Weigh the given amount of Silver Solution from part A andkeep in water bath at 70° C. Add Carbopol to Silver Solution withconstant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 8.43 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min.3. Tackiness Tacky Batch No. SG/011 Formulation: Part A: Distilled Water  98 g Carbopol 0.76 g TEA 0.56 g Part B: Distilled Water  3.0 gPropylene glycol  5.0 g TEA  1.4 g

Procedure: Weigh the given amount of Distilled Water from Part A andkeep in water bath at 70° C. Add Carbopol to Distilled Water Solutionwith constant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results: 1. pH 8.05 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min.3. Tackiness Very Tacky Batch No. SG1012 Formulation: Part A Distilledwater   98 g Carbopol 0.76 g TEA 0.34 g Part B Distilled Water 3.00 gPropylene glycol 5.00 g TEA 0.64 g

Procedure: Weigh the given amount of Distilled Water from Part A andkeep in water bath at 70° C. Add Carbopol to Distilled Water Solutionwith constant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 6.35 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min.3. Tackiness Very Tacky Batch No. SG/013 Formulation: Part A: SilverSolution (32 ppm)   86 g Distilled Water   12 g Carbopol 0.76 g TEA 0.32g Part B: Silver Solution (32 ppm) 3.00 g Propylene glycol 5.00 g TEA0.64 g

Procedure: Weigh the given amount of Silver Solution & Distilled Waterfrom Part A and keep in water bath at 70° C. Add Carbopol to Solutionwith constant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results: 1. pH 6.7 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min.3. Tackiness Very Tacky Batch No. SG/014 Formulation: Part A: SilverSolution (32 ppm)   86 g Distilled Water   12 g Carbopol 0.78 g TEA 0.32gm Part B Silver Solution (32 ppm): 3.00 g Propylene glycol 5.00 g TEA0.64 g

Procedure: Weigh the given amount of Silver Solution & Distilled Waterfrom Part A and keep in water bath at 70° C. Add Carbopol to Solutionwith constant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 6.6 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min. 3.Tackiness Very Tacky Batch No. SG/015 Formulation: Part A SilverSolution (32 ppm)   86 g Distilled Water   12 g Carbopol 0.68 g TEA 0.40g Part B Silver Solution (32 ppm):  5.0 g Propylene glycol  7.0 g TEA 0.6 g

Procedure: Weigh the given amount of Silver Solution & Distilled Waterfrom Part A and keep in water bath at 70° C. Add Carbopol to Solutionwith constant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 6.72 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min 3.Tackiness Tacky Batch No. SG/016 Formulation: Part A: Silver Solution(32 ppm)   86 g Distilled Water   12 g Carbopol 0.64 g TEA 0.40 g PartB: Silver Solution (32 ppm)  5.0 g Propylene glycol  7.0 g TEA  0.6 g

Procedure: Weigh the given amount of Silver Solution & Distilled Waterfrom Part A and keep in water bath at 70° C. Add Carbopol to Solutionwith constant stirring to avoid lumps. Add TEA to it at 70° C. after 20mins. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results: 1. pH 6.87 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min3. Tackiness Tacky Batch No. SG/017 Formulation: Part A: Silver Solution(32 ppm)   86 g Distilled Water   12 g Carbopol 0.62 g TEA  0.4 g PartB: Silver Solution (32 ppm)  5.0 g Propylene glycol  7.0 g TEA  0.6 g

Procedure: Weigh the given amount of Silver Solution & Distilled Waterfrom Part A and keep in water bath at 70° C. Add Carbopol to Solutionwith constant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results: 1. pH 7.05 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min3. Tackiness Tacky Batch No. SG/018 Formulation: Part A: Silver Solution(32 ppm)   86 g Distilled Water   12 g Carbopol 0.58 g TEA  0.4 g PartB: Silver Solution (32 ppm)  5.0 g Propylene glycol  7.0 g TEA  0.6 g

Procedure: Weigh the given amount of Silver Solution & Distilled Waterfrom Part A and keep in water bath at 70° C. Add Carbopol to Solutionwith constant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 7.40 2. Flow-ability 90° C. => >5 min. 45° C. => >5 min 3.Tackiness Smooth Batch No. SG/019 Formulation: Part A: Silver Solution(32 ppm)   86 g Distilled water   12 g Carbopol 0.54 g TEA  0.4 g PartB: Silver Solution (32 ppm)  5.0 g Propylene glycol  7.0 g TEA  0.6 g

Procedure: Weigh the given amount of Silver Solution & Distilled Waterfrom Part A and keep in water bath at 70° C. Add Carbopol to Solutionwith constant stirring to avoid lumps. Add TEA to it at 70° C. after 20minutes. Weigh all the ingredients from Part B and keep in water bath at70° C. for 15-20 min. Add Part B to Part A and stir it for 10-15 min.Cool it to room temperature and analyze.

Results 1. pH 7.65 2. Flow-ability 90° C. => 1 min. 45° C. => 2 min 3.Tackiness Smooth

Remark:

Though the Gel feel has improved consistency is not suitable.

Based on the above results the following instructions for a one kilogrambatch were developed.

Part B Silver Solution  860 gm Distilled water  100 gm Carbopol 5.80 gmTEA 4.00 gm ASAP Solution 50.0 gm Propylene glycol 70.0 gm TEA 6.00 gmYield 1.0 Kg. after adjusting for moisture loss.

Procedure: in a clean sterilized vessel take the required quantity ofDistilled Water & silver solution. Raise the temperature of solution to70° C. with continuous stirring. Start addition of Carbopol in minuteamounts with continuous stirring/homogenization. After all Carbopol hasbeen added continue for 30 minutes. (Adjust time according to batchsize). Then add TEA into the phase A solution.

In a separate vessel mix all ingredients of part B. Raise thetemperature to 70° C. & slowly add part B to Part A. On completehomogenization cool it to room temperature.

Precautions:

Carbopol dispersion must be done using a good homogenizer.

Take a small trial batch when using a new lot of Carbopol.

Minimize Heating time as longer heating leads to more water loss.

Results 1. pH 7.4 2. Flow-ability >5 min. 3. Tackiness Smooth.

This formulation has been readily scaled up to 10 kgs. in a pilot plant.No problems were encountered during scale up. Deaeration by vacuumapplication is recommended to remove entrapped air and ensure uniformfilling.

This formulation has the following physical and chemicalcharacteristics.

TEST SPECIFICATION RESULTS 1. Appearance Golden yellow PassesTranslucent Gel 2. Odor Odorless Odorless 3. Specific Gravity 1.02 1.024. Flowability At 45° & 90° - At 45° & 90° - More than 5 min. More than5 min. to travel 1 inch to travel 1 inch from the origin from the origin5. Foaming Cap. <10 ml <10 ml 6. Feel/Tackiness 1-Smooth 1-Smooth 7Viscosity RT 30° 32,000 ± 5000 34,000 370 30,000 + 5000 33,500 8. PH 6.5to 8.0 7.4 9. Freeze & Thaw To pass SOP 1–10 Compares with Original 10.Optimum 22 ppm - 400 +/− 20 nm. 400 nm. ** Wavelength 32 ppm - 450 +/−20 nm. 450 nm. ** (X Max) 11. Light Exposure No further discolorationPasses. 12. Compatibility No discoloration of Ref Table 3 product Ireaction with containers. 13. Moisture Donation — 10.27% 14. MoistureUptake — 80%

Microbiological Evaluation

It is reasonable to assume that the silver colloid hydrogel hasmicrobiological proprieties similar to the original silver colloid whichhas been extensively tested as demonstrated above. However, the additionof the hydrophilic polymer to produce the gel might directly interferewith the microbial properties of the silver or might so inhibitdiffusion of the silver that effectiveness is decreased. Therefore,microbiological tests similar to those carried out on the silver colloidsolution were also performed on the silver colloid hydrogel.

Initially, the hydrogel was tested to determine whether the compositionwas self-sterilizing. The following protocol was followed:

Flasks of 100 ml sterile Fluid Thioglycollate Medium (Anaerobicbacteria), sterile Soya bean Casein Digest Medium (Aerobic bacteria),and Potato Dextrose Broth (Fungi) were obtained Samples of about 100 mgof gel to be tested were aseptically transferred into sets of flask. Oneset was incubated at 37° C. and another set was incubated at roomtemperature for one week. After that time the flasks were inspected andshowed no turbidity or sign of microbial growth. Because the gel samplehad not been manufactured under sterile conditions, it can be concludedthat the composition is self-sterilizing. The 100 mg of gel used foreach test This corresponds to 2.2 μg in 100 ml medium or 0.02214 or0.032 μg of silver per ml of medium. At this concentration silver wouldnot have antimicrobial activity and hence false negative results can beeliminated.

A variety of test organisms were then used to compare the zone ofinhibition attained with either 22 or 32 ppm silver solution or 22 or 32ppm silver gel made as described above. Aliquots of 0.1 ml or activelygrowing 18 hr cultures of each microorganism (approximately 10⁸ CFU/ml)were spread on sterile nutrient agar plates. A 10 mm diameter hole waspunched in each inoculated plate with a cork borer. a test amount of(0/2-0.3 g) of the product was placed into each hole, and the plate wasincubated for 24 hr. After that time the plates were inspected and thefollowing zones of inhibition (total diameter of each zone) weremeasured.

Silver Solution Silver Gel Culture 22 ppm 32 ppm 22 ppm 32 ppm E. coil14 mm 14 mm 12 mm 13 mm Ps. Aeruginosa 21 mm 22 mm 21 mm 20 mm B.subtilis 15 mm 16 mm 14 mm 14 mm MRSA 1 17 mm 18 mm 16 mm 17 mm MRSA 216 mm 17 mm 16 mm 17 mm S. aureus 14 mm 14.5 mm   15 mm 15 mm ATCC 6538P S. pyogenes 16 mm 18 mm 16 mm 18 mm S. typhi 17 mm 16 mm 16 mm 16 mmSh. flexneri 20 mm 21 mm 20 mm 21 mm K. pneumoniae 17 mm 18 mm 18 mm 18mm C. diptheriae 16 mm 18 mm 16 mm 17 mm C. albicans 39 mm 40 mm 39 mm40 mm

These results show that the inhibitory effects of the gel areessentially equivalent to those of the silver colloid solution; thisdemonstrates that the gelling polymer does not negatively affect theantimicrobial powers of the silver colloid. Some cultures (S. pyogenes,C. diphtheriae and S. aureus) were also cultured on Blood Agar. Theresults suggested that the silver gel would also be effective on abloody, exuding wounds.

Similar tests were carried out on the same bacterial strains using avariety of antibiotic agents. In some cases the antibiotics were moreeffective than the silver compounds—in others they were much lesseffective. This demonstrates that the strains used were not weakened or“push-over” strains.

Gram Positive Bacteria

S. B. Antibiotic Conc. aureus MRSA 1 MRSA 2 subtilis Ampicillin 200 mcg Clear 15 mm 18 mm 16 mm Cefotaxime 30 mcg 26 mm No -Clear  12 mminhibition Cephalexin 30 mcg Clear 1.0 mm  0.8 mm  Clear Ciprofloxacin 5 mcg 28 mm 14 mm 14 mm 20 mm Cloxacillin  1 mcg Clear Clear 13 mm 18mm Co- 25 mcg Clear No No 15 mm Trimoxazole inhibition inhibitionGentamycin 10 mcg Clear No 11 mm 18 mm inhibition Lincomycin  2 mcgClear Clear Clear 18 mm Ofloxacin  5 mcg Clear 15 mm 16 mm 22 mmPeflofloxacin 10 mcg 30 mm 11 mm 13 mm 21 mm Roxythromycin 15 mcg Clear1.0 mm  12 mm 20 mm Tetracyclin 30 mcg 34 mm No 0.7 mm  19 mm inhibition

Gram Negative Bacteria

K. pneu- S. Ps. Antibiotics Conc. E. coli moniae typhi aeruginosaAmikacin 30 mcg Clear 18 mm Clear 10 mm Ampicillin 200 mcg  23 mm 18 mm20 mm 13 mm Cefotaxime 30 mcg 21 mm 20 mm 22 mm 19 mm Ceftizoxime 30 mcg18 mm 18 mm 15 mm No inhibition Chloramphenicol 30 mcg 22 mm 21 mm 23 mmNo inhibition Ciprofloxacin  5 mcg 29 mm 22 mm 25 mm 15 mmCo-Trimoxazole 25 mcg 24 mm 19 mm 27 mm Clear Gentamycin 10 mcg Clear 17mm Clear No inhibition Ofloxacin  5 mcg  Clear- 29 mm clear 15 mmPefloxacin 10 mcg Clear 25 mm clear 10 mm Piperacillin 100 mcg  22 mm 15mm 16 mm 10 mm Tetracyclin 30 mcg 19 mm 18 mm 16 mm No inhibition

Hand Scrub Test

Since the hydrogel has the ability to increase the adherence of silverto skin surfaces, effectiveness of the gel as a hand scrub wasevaluated. For this test a one inch square of a volunteers hand wasmarked and then scrubbed with about 1 g of the gel. A control area wasscrubbed with sterile distilled water. The areas were swabbed and theswab streaked on nutrient agar. The swabbing was repeated every hour forfour hours. The streaked plates were incubated for 24 hr at 37° C. andthe results evaluated.

As shown in the following table, the control swabs grew so many bacteriaas to be Too Numerous To Count (TNTC). The areas treated with silver gelremained essentially sterile for three hours and showed only slightgrowth at four hours. This should provide superior results for healthcare workers who need to sterilize the surface of their hands withoutusing harsh or irrigating compounds.

Time Control 22 ppm 32 ppm 0 hr TNTC No growth No growth 1 hr TNTC Nogrowth No growth 2 hr TNTC No growth No growth 3 hr TNTC No growth Nogrowth 4 hr TNTC 3 Cfu 2 Cfu

Although hydrogels show exceptional wound healing properties, a drawbackof the typical hydrogel is that microorganisms are often able to migratethrough the matrix. Thus, if a would is covered by hydrogel and one areaof the wound becomes infected, the infectious organisms may be able totravel through the hydrogel an infect other regions. This possibilitywas tested by using a strip of hydrogel to bridge separated regions on anutrient agar plate. Each agar plate was separated into two regions byremoving a 2 cm strip of agar along a diameter of the plate. This gapwas bridged by a 1.5 cm wide strip of hydrogel that overlapped onto theagar by about 5 mm at either end. One side of the plate was theninoculated with about 0.5 ml of culture and the plate was incubated tosee if the microorganisms could cross the hydrogel “bridge.” The resultsshow that silver hydrogel completely prevented migration.

Culture Zone of Inoculation Zone of Migration E. coil Heavy Growth NoGrowth B. subtilis Heavy Growth No Growth MRSA 1 Heavy Growth No GrowthPs. aeruginosa Heavy Growth No Growth Hydrogel control Heavy GrowthGrowth

From the results shown above a prototype gel formula was selected andthe variations are suggested in the following examples.

Example 1

For a 1 Kg batch of gel take components of Part A and Part B as givenbelow:

Part A Inventive silver colloid 32 ppm 860 g Distilled Water 100 gCarbapol 6.8 g Triethanolamine 4.0 g Part B nventive silver colloid 32ppm 50 g Propylene Glycol 70 g Triethanolamine 6.0 g

First take required amount of distilled water and silver solution in astirrer and start stirring. Slowly add in Carbapol (Noveon, USA).Stirring should be sufficiently vigorous to dispense the Carbopol andavoid formation of lumps. Temperature should be maintained between60-70° C. during stirring.

Mix all ingredients of Part B in a beaker. Heat to 70° C. and add toPart A under vigorous stirring. Continue mixing and cool to roomtemperature. Check yield of batch. It should be approx. 1000 gm. Thetriethanolamine causes the Carbopol to gel.

Example 2

Prepare all ingredients as in Example 1 including the addition of 1%collagen. This will give a gel with both antimicrobial as well asbenefits of collagen which has scaffolding type of wound healingacceleration.

Example 3

Prepare all ingredients as in Example 1 but including the addition ofAloe vera (powder or solution) in the range of 1-5%. This will conferadditional wound healing properties.

Example 4

Prepare all ingredients as in Example 1 with the addition of 1-10% byweight Maltodextrin. This will provide a gel formulation whichstimulates wound granulation.

Summary of Silver Hydrogel Results

It was possible to prepare Carbopol based gels using inventive silvercolloid solutions of 22 ppm & 32 ppm. The gel thus prepared have manyadvantage over their solution counterparts by virtue of their capacityto remain in place while retaining the properties of the parent silversolutions. The amorphous hydrogel nature of the medicament confers theadvantage of moist wound healing acceleration and also limiting theseverity of burns wound by limiting the thermal shock. Moreover, theactive agent, colloidal silver solution has been tested on a cell linein an earlier study and found to be non-cytotoxic.

A thorough physico-chemical evaluation of the gel has been done withvarious batches and series of series of detailed methods were preparedto standardize and control product and processes during manufacture.

Microbiological studies were carried out in depth and show that the gelretains its bactericidal nature. Silver migration studies have beensimulated and conclusively demonstrate that the gel can deliver silverover a period of time to the wound. The formulation design will also notallow microbe migration inside from outside and vice versa.

These tests demonstrate that hypothetical evaluation of the silverhydrogel on the basis of a publication (Journal of Wound Care Vol 12, No8 SEPT 2003) where alternative silver based dressings were evaluatedwith points given for:

1. Antimicrobial zone of inhibition;

2. Microbial challenge test;

3. Microbial transmission test; and

4. Silver content of dressings.

In the first test the silver hydrogel would be placed in group B and inall three remaining tests the silver hydrogel would score in group Agiving it a total points of 20, on par with Calgitrol Ag & Acticot,commercial products which scored the highest in this evaluation.

The antibacterial and antiviral properties of the colloidal silversolution open up several significant uses for the silver hydrogel beyonda wound dressing. As demonstrated above the hydrogel is an idealantibacterial hand scrub. In addition, the nonirritating character ofthe silver colloid and the hydrogel make the combination an idealpersonal lubricant for male or female sexual use with or without condomsor diaphragms where the combination would combat bacteria, fungi (notethe effectiveness on Candida albicans) and dangerous virus such as HIVand disinfect reusable barriers such as diaphragms. Since the hydrogelcontains little if any oil, it has no harmful effects on condoms ordiaphragms, unlike certain other personal lubricants.

The following claims are thus to be understood to include what isspecifically illustrated and described above, what is conceptuallyequivalent, what can be obviously substituted and also what essentiallyincorporates the essential idea of the invention. Those skilled in theart will appreciate that various adaptations and modifications of thejust-described preferred embodiment can be configured without departingfrom the scope of the invention. The illustrated embodiment has been setforth only for the purposes of example and that should not be taken aslimiting the invention. Therefore, it is to be understood that, withinthe scope of the appended claims, the invention may be practiced otherthan as specifically described herein.

We claim:
 1. A hydrogel composition comprising a hydrophilic acrylicacid copolymers dissolved in a composition of silver in water having atotal concentration of silver of between about 5 and 40 parts permillion, wherein said silver is in the form of a stable and colorlesscolloidal suspension of silver particles having an interior of metallicsilver and an exterior surface of ionic silver oxide, wherein at least75% of the silver particles have diameters between 0.005 micrometers and0.015 micrometers, wherein said silver particles are made from a silverelectrode in an electrochemical cell, and wherein the compositionmanifests antimicrobial properties.
 2. The composition according toclaim 1, wherein at least 90% of the colloidal silver particles havediameters between 0.005 micrometers and 0.015 micrometers.
 3. Thecomposition according to claim 2, wherein at least 95% of the colloidalsilver particles have diameters between 0.005 micrometers and 0.015micrometers.
 4. The composition according to claim 1 further comprisinghydrogen peroxide.
 5. The composition according to claim 4, wherein thehydrogen peroxide concentration is between about 1% weight/vol and about3.0% weight/vol.
 6. The composition according to claim 1, wherein thecomposition manifests antimicrobial properties against microbes selectedfrom the group consisting of Bacillus anthracis, Bacillus subtilis,Candida albicans, Mycobacteria bovis, Mycobacteria tuberculosis,Pseudomonas aeruginosa, Salmonella choleraesius, Staphylococcus aureus,Trichomonas vaginalis, and Yersinia pestis.
 7. The composition accordingto claim 6, wherein Staphylococcus aureus is a methicillin-resistantstrain.
 8. The composition according to claim 1, wherein the compositionmanifests antimicrobial properties against microbes associated withdiseases selected from the group consisting of malaria, fungalinfections of the skin, bacterial infections of the skin, vaginalinfections, urinary tract infections, tonsillitis, pelvic inflammatorydisease, pharyngitis, gonorrhea, conjunctivitis, otitis, respiratorytract infections, and nasal infections.
 9. The composition according toclaim 1, further comprising additives to enhance physicalcharacteristics of the hydrogel and/or enhance wound healing.
 10. Thecomposition according to claim 9, wherein the additives are selectedfrom the group consisting of vitamins, amino acids, growth factors,maltodextrin, aloe vera and anesthetics.
 11. The composition accordingto claim 1, further comprising additional antimicrobial agents.
 12. Thecomposition according to claim 11, wherein the additional antimicrobialagents are selected from the group consisting of organic acids,alcohols, organic disinfectants, chlorinated phenolics, chlorhexidine,biguanides, surfactants, aldehydes, halogen disinfectants andoxygenating disinfectants.